Mitogenic oxygenase regulators

ABSTRACT

The present invention relates to new genes encoding for the production of novel nox enzyme proteins involved in generation of reactive oxygen intermediates that affect cell division. The present invention also provides vectors containing these genes, cells transfected with these vectors, antibodies raised against these novel proteins, kits for detection, localization and measurement of these genes and proteins, and methods to determine the activity of drugs to affect the activity of the proteins of the present invention.

BACKGROUND OF THE INVENTION

[0001] Reactive oxygen intermediates (ROI) are cytotoxic and mutagenic. ROIs modify and damage critical biomolecules including DNA and lipids. The are partial reduction products of oxygen: 1 electron reduces O₂ to form superoxide (O₂ ^(−), and) 2 electrons reduce O₂ to form hydrogen peroxide (H₂O₂). The cytotoxic property of ROI is exploited by phagocytes, which generate large amounts of superoxide and hydrogen peroxide as part of their armory of bactericidal mechanisms. ROI have been considered an accidental byproduct of metabolism, particularly mitochondrial respiration. Recent studies give evidence for regulated enzymatic generation of O₂ ⁻ and its conversion to H₂O₂ in a variety of cells. The conversion of O₂ ⁻ to H₂O₂ can also occur spontaneously, but is markedly accelerated by superoxide dismutase (SOD). Exposure of cells to platelet derived growth factor and epidermal growth factor induces the production of H₂O₂, which activates components of signaling pathways including p42/p44 MAPK and tyrosine phosphroylation.

[0002] Several biological systems generate reactive oxygen. Exposure of neutrophils to bacteria or to various soluble mediators such as formyl-Met-Leu-Phe or phorbol esters activates a massive consumption of oxygen, termed the respiratory burst, to initially generate superoxide, with secondary generation of H₂O₂, HOCl and hydroxyl radical. The enzyme responsible for this oxygen consumption is the respiratory burst oxidase (nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) oxidase).

[0003] There is also growing evidence for the generation of ROI by non-phagocytic cells, particularly in situations related to cell proliferation. Significant generation of H₂O₂, O₂ ⁻, or both have been noted in some cell types. Fibroblasts and human endothelial cells show increased release of superoxide in response to cytokines such as interleukin-1 or tumor necrosis factor (TNF) (Meier et al. (1989) Biochem J. 263, 539-545.; Matsubara et al. (1986) J. Immun. 137, 3295-3298). Ras-transformed fibroblasts show increased superoxide release compared with control fibroblasts (Irani, et al. (1997) Science 275, 1649-1652). Rat vascular smooth muscle cells show increased H₂O₂ release in response to PDGF (Sundaresan et al. (1995) Science 270, 296-299) and angiotensin II (Griendling et al. (1994) Circ. Res. 74, 1141-1148; Fukui et al. (1997) Circ. Res. 80, 45-51; Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317-23321), and H₂O₂ in these cells is associated with increased proliferation rate. H₂O₂ in the transformed fibroblasts and in vascular smooth muscle cells is associated with an increased proliferation rate. The occurrence of ROI in a variety of cell types is summarized in Table 1 (adapted from Burdon, R. (1995) Free Radical Biol. Med. 18, 775-794). TABLE 1 Superoxide Hydrogen Peroxide human fibroblasts Balb/3T3 cells human endothelial cells rat pancreatic islet cells human/rat smooth muscle cells murine keratinocytes human fat cells rabbit chondrocytes human osteocytes human tumor cells BHK-21 cells fat cells, 3T3 L1 cells human colonic epithelial cells

[0004] ROI generated by neutrophils have a cytotoxic function. While ROI are normally directed at the invading microbe, ROI can also induce tissue damage (e.g., in inflammatory conditions such as arthritis, shock, lung disease, and inflammatory bowel disease) or may be involved in tumor initiation or promotion, due to damaging effects on DNA. Nathan (Szatrowski et al. (1991) Canc. Res. 51, 794-798) proposed that the generation of ROI in tumor cells may contribute to the hypermutability seen in tumors, and may therefore contribute to tumor heterogeneity, invasion and metastasis.

[0005] In addition to cytotoxic and mutagenic roles, ROI have ideal properties as signal molecules: 1) they are generated in a controlled manner in response to upstream signals; 2) the signal can be terminated by rapid metabolism of O₂ ⁻ and H₂O₂ by SOD and catalase/peroxidases; 3) they elicit downstream effects on target molecules, e.g., redox-sensitive regulatory proteins such as NFκ-B and AP-1 (Schreck et al. (1991) EMBO J. 10, 2247-2258; Schmidt et al. (1995) Chemistry & Biology 2, 13-22). Oxidants such as O₂ ⁻ and H₂O₂ have a relatively well defined signaling role in bacteria, operating via the SoxI/II regulon to regulate transcription.

[0006] ROI appear to have a direct role in regulating cell division, and may function as mitogenic signals in pathological conditions related to growth. These conditions include cancer and cardiovascular disease. O₂ ⁻ is generated in endothelial cells in response to cytokines, and might play a role in angiogenesis (Matsubara et al. (1986) J. Immun. 137, 3295-3298). O₂ ⁻ and H₂O₂ are also proposed to function as “life-signals”, preventing cells from undergoing apoptosis (Matsubara et al. (1986) J. Immun. 137, 3295-3298). As discussed above, many cells respond to growth factors (e.g., platelet derived growth factor (PDGF), epidermal derived growth factor (EGF), angiotensin II, and various cytokines) with both increased production of O₂ ⁻/H₂O₂ and increased proliferation. Inhibition of ROI generation prevents the mitogenic response. Exposure to exogenously generated O₂ ⁻ and H₂O₂ results in an increase in cell proliferation. A partial list of responsive cell types is shown below in Table 2 (adapted from Burdon, R. (1995) Free Radical Biol. Med. 18, 775-794). TABLE 2 Superoxide Hydrogen peroxide human, hamster fibroblasts mouse osteoblastic cells Balb/3T3 cells Balb/3T3 cells human histiocytic leukemia rat, hamster fibroblasts mouse epidermal cells human smooth muscle cells rat colonic epithelial cells rat vascular smooth muscle cells rat vascular smooth muscle cells

[0007] While non-transformed cells can respond to growth factors and cytokines with the production of ROI, tumor cells appear to produce ROI in an uncontrolled manner. A series of human tumor cells produced large amounts of hydrogen peroxide compared with non-tumor cells (Szatrowski et al. (1991) Canc. Res. 51, 794-798). Ras-transformed NIH 3T3 cells generated elevated amounts of superoxide, and inhibition of superoxide generation by several mechanisms resulted in a reversion to a “normal” growth phenotype.

[0008] O₂ ⁻ has been implicated in maintenance of the transformed phenotype in cancer cells including melanoma, breast carcinoma, fibrosarcoma, and virally transformed tumor cells. Decreased levels of the manganese form of SOD (MnSOD) have been measured in cancer cells and in vitro-transformed cell lines, predicting increased O₂ ⁻ levels (Burdon, R. (1995) Free Radical Biol. Med. 18, 775-794). MnSOD is encoded on chromosome 6q25 which is very often lost in melanoma. Overexpression of MnSOD in melanoma and other cancer cells (Church et al. (1993) Proc. of Natl. Acad. Sci. 90, 3113-3117; Fernandez-Pol et al. (1982) Canc. Res. 42, 609-617; Yan et al. (1996) Canc. Res. 56, 2864-2871) resulted in suppression of the transformed phenotype.

[0009] ROI are implicated in the growth of vascular smooth muscle associated with hypertension, atherosclerosis, and restenosis after angioplasty. O₂ ⁻ generation is seen in rabbit aortic adventitia (Pagano et al. (1997) Proc. Natl. Acad. Sci. 94, 14483-14488). Vascular endothelial cells release O₂ ⁻ in response to cytokines (Matsubara et al. (1986) J. Immun. 137, 3295-3298). O₂ ⁻ is generated by aortic smooth muscle cells in culture, and increased O₂ ⁻ generation is stimulated by angiotensin II which also induces cell hypertrophy. In a rat model system, infusion of angiotensin II leads to hypertension as well as increased O₂ ⁻ generation in subsequently isolated aortic tissue (Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317-23321.; Yu et al. (1997) J. Biol. Chem. 272, 27288-27294). Intravenous infusion of a form of SOD that localizes to the vasculature or an infusion of an O₂ ⁻ scavenger prevented angiotensin II induced hypertension and inhibited ROI generation (Fukui et al. (1997) Circ. Res. 80, 45-51).

[0010] The neutrophil NADPH oxidase, also known as phagocyte respiratory burst oxidase, provides a paradigm for the study of the specialized enzymatic ROI-generating system. This extensively studied enzyme oxidizes NADPH and reduces oxygen to form O₂ ⁻. NADPH oxidase consists of multiple proteins and is regulated by assembly of cytosolic and membrane components. The catalytic moiety consists of flavocytochrome b₅₅₈, an integral plasma membrane enzyme comprised of two components: gp91phox (gp refers to glycoprotein; phox is an abbreviation of the words phagocyte and oxidase) and p22phox (p refers to protein). gp91phox contains 1 flavin adenine dinucleotide (FAD) and 2 hemes as well as the NADPH binding site. p22phox has a C-terminal proline-rich sequence which serves as a binding site for cytosolic regulatory proteins. The two cytochrome subunits, gp91phox and p22phox appear to stabilize one another, since the genetic absence of either subunit, as in the inherited disorder chronic granulomatous disease (CGD), results in the absence of the partner subunit (Yu et al. (1997) J. Biol. Chem. 272, 27288-27294). Essential cytosolic proteins include p47phox, p67phox and the small GTPase Rac, of which there are two isoforms. p47phox and p67phox both contain SH₃ regions and proline-rich regions which participate in protein interactions governing assembly of the oxidase components during activation. The neutrophil enzyme is regulated in response to bacterial phagocytosis or chemotactic signals by phosphorylation of p47phox, and perhaps other components, as well as by guanine nucleotide exchange to activate the GTP-binding protein Rac.

[0011] The origin of ROI in non-phagocytic tissues is unproven, but the occurrence of phagocyte oxidase components has been evaluated in several systems by immunochemical methods, Northern blots and reverse transcriptase-polymerase chain reaction (RT-PCR). The message for p22phox is expressed widely, as is that for Rac1. Several cell types that are capable of O₂ ⁻ generation have been demonstrated to contain all of the phox components including gp91phox, as summarized below in Table 3. These cell types include endothelial cells, aortic adventitia and lymphocytes. TABLE 3 Tissue gp91phox p22phox p47phox p67phox neutrophil   +^(1,2)   +^(1,2) +^(1,2) +^(1,2) aortic adventitia +¹ +¹ +¹   +¹   lymphocytes +² +² +^(1,2) +^(1,2) endothelial cells +² +² +^(1,2) +^(1,2) glomerular mesangial −   +^(1,2) +^(1,2) +^(1,2) cells fibroblasts − +² +^(1,2) +²   aortic sm. muscle −   +^(1,2) ? ?

[0012] However, a distinctly different pattern is seen in several other cell types shown in Table 3 including glomerular mesangial cells, rat aortic smooth muscle and fibroblasts. In these cells, expression of gp91phox is absent while p22phox and in some cases cytosolic phox components have been demonstrated to be present. Since gp91phox and p22phox stabilize one another in the neutrophil, there has been much speculation that some molecule, possibly related to gp91phox, accounts for ROI generation in glomerular mesangial cells, rat aortic smooth muscle and fibroblasts (Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317-23321). Investigation of fibroblasts from a patient with a genetic absence of gp91phox provides proof that the gp91phox subunit is not involved in ROI generation in these cells (Emmendorffer et al. (1993) Eur. J. Haematol. 51, 223-227). Depletion of p22phox from vascular smooth muscle using an antisense approach indicated that this subunit participates in ROI generation in these cells, despite the absence of detectable gp91phox (Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317-23321). At this time the molecular candidates possibly related to gp91phox and involved in ROI generation in these cells are unknown.

[0013] Accordingly, what is needed is the identity of the proteins involved in ROI generation, particularly in non-phagocytic tissues and cells. What is also needed are the nucleotide sequences encoding for these proteins, and the primary sequences of the proteins themselves. Also needed are vectors designed to include nucleotides encoding for these proteins. Probes and PCR primers derived from the nucleotide sequence are needed to detect, localize and measure nucleotide sequences, including mRNA, involved in the synthesis of these proteins. In addition, what is needed is a means to transfect cells with these vectors. What is also needed are expression systems for production of these molecules. Also needed are antibodies directed against these molecules for a variety of uses including localization, detection, measurement and passive immunization.

SUMMARY OF THE INVENTION

[0014] The present invention solves the problems described above by providing a novel family of nucleotide sequences and proteins, termed Nox proteins, encoded by these nucleotide sequences. In particular the present invention provides compositions comprising the nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, and conservative substitutions and fragments thereof, wherein SEQ ID NO:1 and fragments thereof code for the expression of the protein comprising SEQ ID NO:2 and fragments thereof; and SEQ ID NO:3 and fragments thereof code for the expression of the protein comprising SEQ ID NO:4 and fragments thereof. SEQ ID NO:5 is the promoter sequence for Nox 1. While not wanting to be bound by the following statement, it is believed that these Nox proteins, SEQ ID NOs: 2 and 4, and fragments thereof, are involved in ROI production. The present invention also provides vectors containing these nucleotide sequences, cells transfected with these vectors which produce the proteins comprising SEQ ID NO:2 and SEQ ID NO:4, or fragments thereof, and antibodies to these proteins and fragments thereof. The present invention also provides methods for stimulating cellular proliferation by administering vectors encoded for production of the proteins comprising SEQ ID NO:2 or SEQ ID NO:4, and fragments thereof. The present invention further provides methods for stimulating cellular proliferation by administering the proteins comprising SEQ ID NO:2 or SEQ ID NO:4, and fragments thereof. The nucleotides and antibodies of the present invention are useful for the detection, localization and measurement of the nucleic acids encoding for the production of the proteins of the present invention, and also for the detection, localization and measurement of the proteins of the present invention. These nucleotides and antibodies can be combined with other reagents in kits for the purposes of detection, localization and measurement.

[0015] Most particularly, the present invention involves a method for regulation of cell division or cell proliferation by modifying the activity or expression of the proteins described as SEQ ID NO:2 or SEQ ID NO:4, or fragments thereof. These proteins, in their naturally occurring or expressed forms, are expected to be useful in drug development, for example for screening of chemical and drug libraries by observing inhibition of the activity of these enzymes. Such chemicals and drugs would likely be useful as treatments for cancer, prostatic hypertrophy, benign prostatic hypertrophy, hypertension, atherosclerosis and many other disorders involving abnormal cell growth or proliferation as described below. The entire expressed protein may be useful in these assays. Portions of the molecule which may be targets for inhibition or modification include, but are not limited to, the binding site for pyridine nucleotides (NADPH or NADH), the flavoprotein domain (approximately the C-terminal 265 amino acids), and/or the binding or catalytic site for flavin adenine dinucleotide (FAD).

[0016] The present invention further comprises the creation of reporter-promoter constructs for use in assays to measure the activity of compounds. The method of the present invention may additionally be used for the development of drugs or other therapies for the treatment of conditions associated with abnormal growth including, but not limited to, cancer, psoriasis, prostatic hypertrophy, benign prostatic hypertrophy, cardiovascular disease, proliferation of vessels, including but not limited to blood vessels and lymphatic vessels, arteriovenous malformation, vascular problems associated with the eye, atherosclerosis, hypertension, and restenosis following angioplasty. The enzymes of the present invention are excellent targets for the development of drugs and other agents which may modulate the activity of these enzymes. It is to be understood that modulation of activity may result in enhanced, diminished or absence of enzymatic activity. Modulation of the activity of these enzymes may be useful in treatment of conditions associated with abnormal growth.

[0017] Drugs which affect the activity of the enzymes represented in SEQ ID NO:2, SEQ ID NO:4, or fragments thereof, may also be combined with other therapeutics in the treatment of specific conditions. For example, these drugs may be combined with angiogenesis inhibitors in the treatment of cancer, with antihypertensives for the treatment of hypertension, and with cholesterol lowering drugs for the treatment of atherosclerosis.

[0018] Accordingly, an object of the present invention is to provide nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production.

[0019] Another object of the present invention is to provide vectors containing these nucleotide sequences, or fragments thereof.

[0020] Yet another object of the present invention is to provide cells transfected with these vectors.

[0021] Still another object of the present invention is to administer cells transfected with these vectors to animals and humans.

[0022] Another object of the present invention is to provide proteins, or fragments thereof, that are involved in ROI production.

[0023] Still another object of the present invention is to provide antibodies, including monoclonal and polyclonal antibodies, or fragments thereof, raised against proteins, or fragments thereof, that are involved in ROI production.

[0024] Another object of the present invention is to administer genes containing nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production, to animals and humans, and also to cells obtained from animals and humans.

[0025] Another object of the present invention is to administer antisense complimentary sequences of genes containing nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production, to animals and humans and also to cells obtained from animals and humans.

[0026] Yet another object of the present invention is to provide a method for stimulating or inhibiting cellular proliferation by administering vectors containing nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production, to animals and humans. It is also an object of the present invention to provide a method for stimulating or inhibiting cellular proliferation by administering vectors containing antisense complimentary sequences of nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production, to animals and humans. These methods of stimulating cellular proliferation are useful for a variety of purposes, including but not limited to, developing animal models of tumor formation, stimulating cellular proliferation of blood marrow cells following chemotherapy or radiation, or in cases of anemia.

[0027] Still another object of the present invention is to provide antibodies useful in immunotherapy against cancers expressing the proteins represented in SEQ ID NO:2, SEQ ID NO:4, or fragments thereof.

[0028] Yet another object of the present invention is to provide nucleotide probes useful for the detection, localization and measurement of nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production.

[0029] Another object of the present invention is to provide antibodies useful for the detection, localization and measurement of nucleotide sequences, or fragments thereof, encoding for the production of proteins, or fragments thereof, that are involved in ROI production.

[0030] Another object of the present invention is to provide kits useful for detection of nucleic acids including the nucleic acids including the nucleic acids represented in SEQ ID NO:1, SEQ ID NO:3, or fragments thereof, that encode for proteins, or fragments thereof, that are involved in ROI production.

[0031] A further object of the present invention is to provide kits useful for detection of nucleic acids including nucleic acids represented in SEQ ID NO:5, or fragments thereof, representing the promoter region of Nox 1.

[0032] Still another object of the present invention is to provide kits useful for the localization of nucleic acids including the nucleic acids represented in SEQ ID NO:1, SEQ ID NO:3, or fragments thereof, that encode for proteins, or fragments thereof that are involved in ROI production.

[0033] Yet another object of the present invention is to provide kits useful for the localization of nucleic acids including the nucleic acids represented in SEQ ID NO:5 or fragments thereof, representing the promoter region of Nox 1.

[0034] Another object of the present invention is to provide kits useful for detection of proteins, including the proteins represented in SEQ ID NO:2 and SEQ ID NO:4, or fragments thereof, that are involved in ROI production.

[0035] Yet another object of the present invention is to provide kits useful for detection and measurement of proteins, including the proteins represented in SEQ ID NO:2 and SEQ ID NO:4, or fragments thereof, that are involved in ROI production.

[0036] Still another object of the present invention is to provide kits useful for localization of proteins, including the proteins represented in SEQ ID NO:2 and SEQ ID NO:4, or fragments thereof, that are involved in ROI production.

[0037] Yet another object of the present invention is to provide kits useful for the detection, measurement or localization of nucleic acids, or fragments thereof, encoding for proteins, or fragments thereof, that are involved in ROI production, for use in diagnosis and prognosis of abnormal cellular proliferation related to ROI production.

[0038] Another object of the present invention is to provide kits useful for the detection, measurement or localization of proteins, or fragments thereof, that are involved in ROI production, for use in diagnosis and prognosis of abnormal cellular proliferation related to ROI production.

[0039] A further object of the present invention is to use the proteins represented in SEQ ID NO:2 and SEQ ID NO:4, or fragments thereof, to screen for drugs that regulate the cellular levels or activity of proteins in the Nox family.

[0040] These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0041]FIG. 1 is a dendrogram indicating the degree of similarity among this family of proteins, and also includes the related plant enzymes.

[0042] FIGS. 2(a-b) depicts the alignment of the predicted protein sequences of gp91phox, Nox 1, Nox 3, Nox 4 and Nox 5.

[0043]FIG. 3 is a model consistent with the known features of gp91phox.

[0044] FIGS. 4(a-b) depicts tissue expression of Nox 3, Nox 4, and Nox 5 measured by Northern Blot analysis.

[0045]FIG. 5 shows RT-PCR measurement of tissue expression of the Nox family of proteins. RT-PCR was carried out using Nox-specific PCR primers as described herein. The number of cycles was 35, except where indicated (number of cycles in parentheses).

[0046]FIG. 6 (a-b) depicts expression of Nox isoforms in tumor or transformed cell lines; FIG. 6c shows the ratio of expression of gp91phox, Nox 4, and Nox 5 compared with glyceraldehyde-3-phosphate dehydrogenase (G3PDH), obtained from real time PCR results.

[0047]FIG. 7 depicts the creation of a promoter-reporter construct for Nox 1.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention solves the problems described above by providing a novel family of nucleotide sequences, and proteins termed Nox proteins, encoded by these nucleotide sequences. The term “Nox” refers to “NADPH-oxidase.” These novel proteins are part of a larger related family of proteins that generate ROI, including mox proteins (mox is an abbreviation for mitogenic NADPH oxidase), and Duox proteins, (duox is an abbreviation for dual oxidase). In particular, the present invention provides novel compositions comprising the nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, and fragments thereof. SEQ ID NO:1, or fragments thereof, encode for proteins comprising SEQ ID NO:2 or fragments thereof. SEQ ID NO:3, or fragments thereof, encode for proteins comprising SEQ ID NO:4 or fragments thereof. SEQ ID NO:5 is the promoter region for Nox 1.

[0049] The Nox proteins described herein have homology to the gp91phox protein involved in ROI generation, however, the Nox proteins comprise a novel and distinct family of proteins. The Nox proteins included in the present invention have a molecular weight of approximately 65 kDa as determined by reducing gel electrophoresis and are capable of inducing ROI generation in cells. As described in detail below, the Nox proteins of the present invention also function in the regulation of cell growth, and are therefore implicated in diseases involving abnormal cell growth such as cancer. The present invention describes Nox proteins found in humans, however, it is likely that the Nox family of genes/proteins is widely distributed among multicellular organisms.

[0050] In addition to the nucleotide sequences described above, the present invention also provides vectors containing these nucleotide sequences and fragments thereof, cells transfected with these vectors which produce the proteins comprising SEQ ID NO:2, SEQ ID NO:4, and fragments thereof, and antibodies to these proteins and fragments thereof. The present invention also provides methods for stimulating cellular proliferation by administering vectors, or cells containing vectors, encoded for production of the proteins comprising SEQ ID NO:2, SEQ ID NO:4, and fragments thereof. The nucleotides and antibodies of the present invention are useful for the detection, localization and measurement of the nucleic acids encoding for the production of the proteins of the present invention, and also for the detection, localization and measurement of the proteins of the present invention. These nucleotides and antibodies can be combined with other reagents in kits for the purposes of detection, localization and measurement. These kits are useful for diagnosis and prognosis of conditions involving cellular proliferation associated with production of reactive oxygen intermediates.

[0051] The present invention solves the problems described above by providing a composition comprising the nucleotide sequence SEQ ID NO:1 and fragments thereof. The present invention also provides a composition comprising the nucleotide sequence SEQ ID NO:3 and fragments thereof. The present invention additionally provides a composition comprising the nucleotide sequence SEQ ID NO: 5 and fragments thereof.

[0052] The present invention provides a composition comprising the protein SEQ ID NO:2 encoded by the nucleotide sequence SEQ ID NO:1. The present invention additionally provides a composition comprising the protein SEQ ID NO:4 encoded by the nucleotide sequence SEQ ID NO:3.

[0053] The present invention provides a composition comprising the protein SEQ ID NO:2 or fragments thereof, encoded by the nucleotide sequence SEQ ID NO:1 or fragments thereof. The present invention also provides a composition comprising the protein SEQ ID NO:4 or fragments thereof, encoded by the nucleotide sequence SEQ ID NO:3 or fragments thereof.

[0054] The present invention also provides vectors containing the nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, and fragments thereof. The present invention also provides cells transfected with these vectors. In addition, the present invention provides cells stably transfected with the nucleotide sequence SEQ ID NO:1 or fragments thereof. The present invention also provides cells stably transfected with the nucleotide sequence SEQ ID NO:3 or fragments thereof.

[0055] The present invention provides cells stably transfected with the nucleotide sequence SEQ ID NO:1 or fragments thereof, which produce the protein SEQ ID NO:2 or fragments thereof. In addition, the present invention provides cells stably transfected with the nucleotide sequence SEQ ID NO:3 or fragments thereof which produce the protein SEQ ID NO:4 or fragments thereof.

[0056] The present invention provides a method for stimulating growth by administering cells stably transfected with the nucleotide sequence SEQ ID NO:1 which produce the protein SEQ ID NO:2 or fragments thereof. The present invention also provides a method for stimulating growth by administering cells stably transfected with the nucleotide sequence SEQ ID NO:3 or fragments thereof, which produce the protein SEQ ID NO:4 or fragments thereof.

[0057] Specifically, the present invention provides a method for stimulating tumor formation by administering cells stably transfected with the nucleotide sequence SEQ ID NO:1 or fragments thereof, which produce the protein SEQ ID NO:2 or fragments thereof. The present invention also provides a method for stimulating tumor formation by administering cells stably transfected with the nucleotide sequence SEQ ID NO:3 or fragments thereof, which produce the protein SEQ ID NO:4 or fragments thereof.

[0058] The present invention may also be used to develop anti-sense nucleotide sequences to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or fragments thereof. These anti-sense molecules may be used to interfere with translation of nucleotide sequences, such as SEQ ID NO:1, or SEQ ID NO:3, or fragments thereof, that encode respectively, for proteins such as SEQ ID NO:2, SEQ ID NO:4, or fragments thereof. Administration of these anti-sense molecules, or vectors encoding for these anti-sense molecules, to humans and animals, would interfere with production of proteins such as SEQ ID NO:2, SEQ ID NO:4, or fragments thereof, thereby decreasing production of ROIs and inhibiting cellular proliferation. These methods are useful in producing animal models for use in study of tumor development and vascular growth, and for study of the efficacy of treatments for affecting tumor and vascular growth in vivo.

[0059] The present invention also provides a method for high throughput screening of drugs and chemicals which modulate the proliferative activity of the enzymes of the present invention, thereby affecting cell division. Combinatorial chemical libraries may be screened for chemicals which modulate the proliferative activity of these enzymes. Drugs and chemicals may be evaluated based on their ability to modulate the enzymatic activity of the expressed or endogenous proteins, including those represented by SEQ ID NO:2, SEQ ID NO:4, or fragments thereof. Endogenous proteins may be obtained from many different tissues or cells, such as colon cells. Drugs may also be evaluated based on their ability to bind to the expressed or endogenous proteins represented by SEQ ID NO:2, SEQ ID NO:4, or fragments thereof. Enzymatic activity may be NADPH- or NADH-dependent superoxide generation catalyzed by the holoprotein. Enzymatic activity may also be NADPH- or NADH-dependent diaphorase activity catalyzed by either the holoprotein or the flavoprotein domain.

[0060] By flavoprotein domain is meant approximately the C-terminal half of the enzymes shown in SEQ ID NO:2, SEQ ID NO:4, or fragments thereof.

[0061] These proteins and fragments thereof have NADPH-dependent reductase activity towards cytochrome c, nitrobluetetrazolium and other dyes. Expressed proteins or fragments thereof can be used for robotic screens of existing combinatorial chemical libraries. While not wanting to be bound by the following statement, it is believed that the NADPH or NADH binding site and the FAD binding site are useful for evaluating the ability of drugs and other compositions to bind to the Nox enzymes or to modulate their enzymatic activity. The use of the holoprotein or the C-terminal half or end regions are preferred for developing a high throughput drug screen.

[0062] The present invention also provides antibodies directed to the proteins SEQ ID NO:2, SEQ ID NO:4, and fragments thereof. The antibodies of the present invention are useful for a variety of purposes including localization, detection and measurement of the proteins SEQ ID NO:2, SEQ ID NO:4, and fragments thereof. The antibodies may be employed in kits to accomplish these purposes. These antibodies may also be linked to cytotoxic agents for selected killing of cells. The term antibody is meant to include any class of antibody such as IgG, IgM and other classes. The term antibody also includes a completely intact antibody and also fragments thereof, including but not limited to Fab fragments and Fab+Fc fragments.

[0063] The present invention also provides the nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, and fragments thereof. These nucleotide sequences are useful for a variety of purposes including localization, detection, and measurement of messenger RNA involved in synthesis of the proteins represented as SEQ ID NO:2, SEQ ID NO:4, and fragments thereof. The present invention also provides the nucleotide sequence for SEQ ID NO:5 and fragments thereof. This nucleotide sequence is useful for a variety of purposes including localization, detection and measurement of messenger RNA involved in synthesis of the Nox family of proteins. These nucleotides may also be used in the construction of labeled probes for the localization, detection, and measurement of nucleic acids such as messenger RNA or alternatively for the isolation of larger nucleotide sequences containing the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or fragments thereof. These nucleotide sequences may be used to isolate homologous strands from other species using techniques known to one of ordinary skill in the art. These nucleotide sequences may also be used to make probes and complementary strands.

[0064] Most particularly, the present invention involves a method for modulation of growth by modifying the proteins represented as SEQ ID NO:2, SEQ ID NO:4, or fragments thereof.

[0065] The term “mitogenic regulators” is used herein to mean any molecule that acts to affect cell division.

[0066] The term “animal” is used herein to mean humans and non-human animals of both sexes.

[0067] The terms “a”, “an” and “the” as used herein are defined to mean one or more and include the plural unless the context is inappropriate.

[0068] “Proteins”, “peptides,” “polypeptides” and “oligopeptides” are chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. The terminal amino acid at one end of the chain (i.e., the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (i.e., the carboxy terminal) has a free carboxyl group. As such, the term “amino terminus” (abbreviated N-terminus) refers to the free alpha-amino group on the amino acid at the amino terminal of the protein, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the protein. Similarly, the term “carboxy terminus” (abbreviated C-terminus) refers to the free carboxyl group on the amino acid at the carboxy terminus of a protein, or to the carboxyl group of an amino acid at any other location within the protein.

[0069] Typically, the amino acids making up a protein are numbered in order, starting at the amino terminal and increasing in the direction toward the carboxy terminal of the protein. Thus, when one amino acid is said to “follow” another, that amino acid is positioned closer to the carboxy terminal of the protein than the preceding amino acid.

[0070] The term “residue” is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a protein by an amide bond. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.

[0071] Furthermore, one of skill in the art will recognize that, as mentioned above, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (less than about 20%, typically less than about 10%, more typically less than about 1%) in an encoded sequence are conservatively modified variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

[0072] 1) Alanine (A), Serine (S), Threonine (T);

[0073] 2) Aspartic acid (D), Glutamic acid (E);

[0074] 3) Asparagine (N), Glutamine (Q);

[0075] 4) Arginine (R), Lysine (K);

[0076] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0077] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0078] When the peptides are relatively short in length (i.e., less than about 50 amino acids), they are often synthesized using standard chemical peptide synthesis techniques. Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the antigenic epitopes described herein. Techniques for solid phase synthesis are known to those skilled in the art.

[0079] Alternatively, the antigenic epitopes described herein are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid sequence that encodes the peptide or protein, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the peptide or protein in a host, isolating the expressed peptide or protein and, if required, renaturing the peptide or protein. Techniques sufficient to guide one of skill through such procedures are found in the literature.

[0080] When several desired protein fragments or peptides are encoded in the nucleotide sequence incorporated into a vector, one of skill in the art will appreciate that the protein fragments or peptides may be separated by a spacer molecule such as, for example, a peptide, consisting of one or more amino acids. Generally, the spacer will have no specific biological activity other than to join the desired protein fragments or peptides together, or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity. Nucleotide sequences encoding for the production of residues which may be useful in purification of the expressed recombinant protein may be built into the vector. Such sequences are known in the art. For example, a nucleotide sequence encoding for a poly histidine sequence may be added to a vector to facilitate purification of the expressed recombinant protein on a nickel column.

[0081] Once expressed, recombinant peptides, polypeptides and proteins can be purified according to standard procedures known to one of ordinary skill in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Substantially pure compositions of about 50 to 99% homogeneity are preferred, and 80 to 95% or greater homogeneity are most preferred for use as therapeutic agents.

[0082] One of skill in the art will recognize that after chemical synthesis, biological expression or purification, the desired proteins, fragments thereof and peptides may possess a conformation substantially different than the native conformations of the proteins, fragments thereof and peptides. In this case, it is often necessary to denature and reduce protein and then to cause the protein to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art.

[0083] The genetic constructs of the present invention include coding sequences for different proteins, fragments thereof, and peptides. The genetic constructs also include epitopes or domains chosen to permit purification or detection of the expressed protein. Such epitopes or domains include DNA sequences encoding the glutathione binding domain from glutathione S-transferase, hexa-histidine, thioredoxin, hemagglutinin antigen, maltose binding protein, and others commonly known to one of skill in the art. The preferred genetic construct includes the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or fragments thereof. It is to be understood that additional or alternative nucleotide sequences may be included in the genetic constructs in order to encode for the following: a) multiple copies of the desired proteins, fragments thereof, or peptides; b) various combinations of the desired proteins, fragments thereof, or peptides; and c) conservative modifications of the desired proteins, fragments thereof, or peptides, and combinations thereof. Preferred proteins include the human Nox 4 protein and human Nox 5 protein shown as SEQ ID NO:2 and SEQ ID NO:4, respectively, and fragments thereof or conservative substitutions thereof.

[0084] The nucleotide sequences of the present invention may also be employed to hybridize to nucleic acids such as DNA or RNA nucleotide sequences under high stringency conditions which permit detection, for example, of alternately spliced messages.

[0085] The genetic construct is expressed in an expression system such as in NIH 3T3 cells using recombinant sequences in a pcDNA-3 vector (Invitrogen, Carlsbad, Calif.) to produce a recombinant protein. Preferred expression systems include but are not limited to Cos-7 cells, insect cells using recombinant baculovirus, and yeast. It is to be understood that other expression systems known to one of skill in the art may be used for expression of the genetic constructs of the present invention. The preferred proteins of the present invention are the sequences referred to herein as human Nox 4 and human Nox 5 or fragments thereof which have the amino acid sequences set forth in SEQ ID NO:2 and SEQ ID NO:4, respectively, or an amino acid sequence having amino acid substitutions as defined in the definitions that do not significantly alter the function of the recombinant protein in an adverse manner.

[0086] Terminology

[0087] It should be understood that some of the terminology used to describe the novel Nox proteins contained herein is different from the terminology in PCT/US99/26592, U.S. non-provisional application Ser. No. 09/437,568 and U.S. provisional application serial Nos. 60/251,364, 60/249,305, and 60/289,172. The terms mox and nox are equivalents. As described herein, the term “human Nox 4” refers to a protein comprising an amino acid sequence as set forth in SEQ ID NO:2, or fragments or conservative substitutions thereof, and encoded by the nucleotide sequence as set forth in SEQ ID NO:1, or fragments or conservative substitutions thereof. As described herein, the term “human Nox 5” refers to a protein comprising an amino acid sequence as set forth in SEQ ID NO:4, or fragments or conservative substitutions thereof, and encoded by the nucleotide sequence as set forth in SEQ ID NO:3, or fragments or conservative substitutions thereof. The promoter for “human Nox 1” refers to a nucleic acid sequence as set forth in SEQ ID NO:5 or fragments or conservative substitutions thereof.

[0088] Construction of the Recombinant Gene

[0089] The desired gene is ligated into a transfer vector, such as pcDNA3, and the recombinants are used to transform host cells such as Cos-7 cells. It is to be understood that different transfer vectors, host cells, and transfection methods may be employed as commonly known to one of ordinary skill in the art. Three desired genes for use in transfection are shown in SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5. For example, lipofectamine-mediated transfection and in vivo homologous recombination was used to introduce the Nox 4 gene (SEQ ID NO:1) into NIH 3T3 cells.

[0090] The synthetic gene is cloned and the recombinant construct containing a Nox gene is produced and grown in confluent monolayer cultures of a Cos-7 cell line. The expressed recombinant protein is then purified, preferably using affinity chromatography techniques, and its purity and specificity determined by known methods.

[0091] A variety of expression systems may be employed for expression of the recombinant protein. Such expression methods include, but are not limited to the following: bacterial expression systems, including those utilizing E. coli and Bacillus subtilis; virus systems; yeast expression systems; cultured insect and mammalian cells; and other expression systems known to one of ordinary skill in the art.

[0092] Transfection of Cells

[0093] It is to be understood that the vectors of the present invention may be transfected into any desired cell or cell line. Both in vivo and in vitro transfection of cells are contemplated as part of the present invention. Preferred cells for transfection include but are not limited to the following: fibroblasts (possibly to enhance wound healing and skin formation), granulocytes (possible benefit to increase function in a compromised immune system as seen in AIDS, and aplastic anemia), muscle cells, neuroblasts, stem cells, bone marrow cells, osteoblasts, B lymphocytes, and T lymphocytes.

[0094] Cells may be transfected with a variety of methods known to one of ordinary skill in the art and include but are not limited to the following: electroporation, gene gun, calcium phosphate, lipofectamine, and fugene, as well as adenoviral transfection systems.

[0095] Host cells transfected with the nucleic acids represented in SEQ ID NO:1, SEQ ID NO:3, or fragments thereof, are used to express the proteins SEQ ID NO:2, SEQ ID NO:4, respectively, or fragments thereof. Host cells transfected with the nucleic acid represented in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 or fragments thereof, are also used as screening assays.

[0096] These expressed proteins are used to raise antibodies. These antibodies may be used for a variety of applications including but not limited to immunotherapy against cancers expressing one of the Nox proteins, and for detection, localization and measurement of the proteins shown in SEQ ID NO:2, SEQ ID NO:4, or fragments thereof.

[0097] Purification and Characterization of the Expressed Protein

[0098] The proteins of the present invention can be expressed as a fusion protein with a poly histidine component, such as a hexa histidine, and purified by binding to a metal affinity column using nickel or cobalt affinity matrices. The protein can also be expressed as a fusion protein with glutathione S-transferase and purified by affinity chromatography using a glutathione agarose matrix. The protein can also be purified by immunoaffinity chromatography by expressing it as a fusion protein, for example with hemagglutinin antigen. The expressed or naturally occurring protein can also be purified by conventional chromatographic and purification methods which include anion and cation exchange chromatography, gel exclusion chromatography, hydroxylapatite chromatography, dye binding chromatography, ammonium sulfate precipitation, precipitation in organic solvents or other techniques commonly known to one of skill in the art.

[0099] Methods of Assessing Activity of Expressed Proteins

[0100] Different methods are available for assessing the activity of the expressed proteins of the present invention, including but not limited to the proteins represented as SEQ ID NO:2, SEQ ID NO:4, conservative substitutions thereof, and fragments thereof.

[0101] 1. Assays of the Holoprotein and Fragments Thereof for Superoxide Generation

[0102] A. General Considerations.

[0103] These assays are useful in assessing efficacy of drugs designed to modulate the activity of the enzymes of the present invention. The holoprotein may be expressed in COS-7 cells, NIH 3T3 cells, insect cells (using baculoviral technology) or other cells using methods known to one of skill in the art. Membrane fractions or purified protein are used for the assay. The assay may require or be augmented by other cellular proteins such as p47phox, p67phox, and Rac1, as well as potentially other unidentified factors (e.g., kinases or other regulatory proteins).

[0104] B. Cytochrome c Reduction.

[0105] NADPH or NADH is used as the reducing substrate, in a concentration of about 100 μM. Reduction of cytochrome c is monitored spectrophotometrically by the increase in absorbance at 550 nm, assuming an extinction coefficient of 21 mM⁻¹cm⁻¹. The assay is performed in the absence and presence of about 10 μg superoxide dismutase. The superoxide-dependent reduction is defined as cytochrome c reduction in the absence of superoxide dismutase minus that in the presence of superoxide dismutase (Uhlinger et al. (1991) J. Biol. Chem. 266, 20990-20997). Acetylated cytochrome c may also be used, since the reduction of acetylated cytochrome c is thought to be exclusively via superoxide.

[0106] C. Nitroblue Tetrazolium Reduction.

[0107] For nitroblue tetrazolium (NBT) reduction, the same general protocol is used, except that NBT is used in place of cytochrome c. In general, about 1 mL of filtered 0.25% nitrotetrazolium blue (Sigma, St. Louis, Mo.) is added in Hanks buffer without or with about 600 Units of superoxide dismutase (Sigma) and samples are incubated at approximately 37° C. The oxidized NBT is clear, while the reduced NBT is blue and insoluble. The insoluble product is collected by centrifugation, and the pellet is re-suspended in about 1 mL of pyridine (Sigma) and heated for about 10 minutes at 100° C. to solubilize the reduced NBT. The concentration of reduced NBT is determined by measuring the absorbance at 510 nm, using an extinction coefficient of 11,000 M⁻¹cm⁻¹. Untreated wells are used to determine cell number.

[0108] D. Luminescence.

[0109] Superoxide generation may also be monitored with a chemiluminescence detection system utilizing lucigenin (bis-N-methylacridinium nitrate, Sigma, St. Louis, Mo.). The sample is mixed with about 100 μM NADPH (Sigma, St. Louis, Mo.) and 10 μM lucigenin (Sigma, St. Louis, Mo.) in a volume of about 150 μL Hanks solution. Luminescence is monitored in a 96-well plate using a LumiCounter (Packard, Downers Grove, Ill.) for 0.5 second per reading at approximately 1 minute intervals for a total of about 5 minutes; the highest stable value in each data set is used for comparisons. As above, superoxide dismutase is added to some samples to prove that the luminescence arises from superoxide. A buffer blank is subtracted from each reading (Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317-23321).

[0110] E. Assays in Intact Cells.

[0111] Assays for superoxide generation may be performed using intact cells, for example, the Nox-transfected NIH 3T3 cells. In principle, any of the above assays can be used to evaluate superoxide generation using intact cells, for example, the Nox-transfected NIH 3T3 cells. NBT reduction is a preferred assay method.

[0112] 2. Assays of Truncated Proteins Comprised of Approximately the C-Terminal 265 Amino Acid Residues

[0113] While not wanting to be bound by the following statement, the truncated protein comprised of approximately the C-terminal 265 amino acid residues is not expected to generate superoxide, and therefore, superoxide dismutase is not added in assays of the truncated protein. Basically, a similar assay is established and the superoxide-independent reduction of NBT, cytochrome c, dichlorophenolindophenol, ferricyanide, or another redox-active dye is examined.

[0114] Nucleotides and Nucleic Acid Probes

[0115] The nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, as well as fragments thereof and PCR primers therefore, may be used, respectively, for localization, detection and measurement of nucleic acids related to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, as well as fragments thereof. The nucleotide sequences SEQ ID NO:1 and SEQ ID NO:3 are also called the human Nox 4 gene and the human Nox 5 gene respectively, in this application. The nucleotide sequence SEQ ID NO:5 is called the Nox 1 promoter sequence in this application.

[0116] The nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, as well as fragments and conservative substitutions thereof, may be used to create probes to isolate larger nucleotide sequences containing the nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, respectively. The nucleotide sequences SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5 as well as fragments thereof and conservative substitutions thereof, may also be used to create probes to identify and isolate Nox proteins in other species.

[0117] The nucleic acids described herein include messenger RNA coding for production of SEQ ID NO:2, SEQ ID NO:4, and fragments and conservative substitutions thereof. Such nucleic acids include but are not limited to cDNA probes. These probes may be labeled in a variety of ways known to one of ordinary skill in the art. Such methods include but are not limited to isotopic and non-isotopic labeling. These probes may be used for in situ hybridization for localization of nucleic acids such as mRNA encoding for SEQ ID NO:2, SEQ ID NO:4, and fragments and conservative substitutions thereof. Localization may be performed using in situ hybridization at both ultrastructural and light microscopic levels of resolution using techniques known to one of ordinary skill in the art.

[0118] These probes may also be employed to detect and quantitate nucleic acids and mRNA levels using techniques known to one of ordinary skill in the art including but not limited to solution hybridization.

[0119] Administration of the Nox Proteins of the Present Invention

[0120] The proteins represented by SEQ ID NO:2, or SEQ ID NO:4, or fragments or conservative substitutions thereof, are combined with a pharmaceutically acceptable carrier or vehicle to produce a pharmaceutical composition and are administered to animals. Such administration may occur for stimulation of growth or cellular proliferation. Administration may also occur for generation of antibodies.

[0121] The terms “pharmaceutically acceptable carrier or pharmaceutically acceptable vehicle” are used herein to mean any liquid including but not limited to water or saline, oil, gel, salve, solvent, diluent, fluid ointment base, liposome, micelle, giant micelle, and the like, which is suitable for use in contact with living animal or human tissue without causing adverse physiological responses, and which does not interact with the other components of the composition in a deleterious manner.

[0122] The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers.

[0123] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.

[0124] Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents commonly used by one of ordinary skill in the art.

[0125] The pharmaceutical composition may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. The pharmaceutical composition of the present invention may be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes.

[0126] The pharmaceutical composition may be stored at temperatures of from about 4° C. to −100° C. The pharmaceutical composition may also be stored in a lyophilized state at different temperatures including room temperature. The pharmaceutical composition may be sterilized through conventional means known to one of ordinary skill in the art. Such means include, but are not limited to filtration, radiation and heat. The pharmaceutical composition of the present invention may also be combined with bacteriostatic agents, such as thimerosal, to inhibit bacterial growth.

[0127] Administration may also occur for the production of polyclonal antibodies using methods known to one of ordinary skill in the art. The preferred animals for antibody production are rabbits and mice. Other animals may be employed for immunization with these proteins or fragments thereof. Such animals include, but are not limited to the following; sheep, horses, pigs, donkeys, cows, monkeys and rodents such as guinea pigs and rats. It is expected that from about 1 to 7 dosages may be required per immunization regimen. Initial injections may range from about 0.1 μg to 1 mg, with a preferred range of about 1 μg to 800 μg, and a more preferred range of from approximately 25 μg to 500 μg. Booster injections may range from 0.1 μg to 1 mg, with a preferred range of approximately 1 μg to 800 μg, and a more preferred range of about 10 μg to 500 μg.

[0128] The volume of administration will vary depending on the route of administration and the size of the recipient. For example, intramuscular injections may range from about 0.1 ml to 1.0 ml.

[0129] Adjuvants

[0130] A variety of adjuvants known to one of ordinary skill in the art may be administered in conjunction with the protein in the pharmaceutical composition for generation of antibodies. Such adjuvants include, but are not limited to the following: polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers; polymer P1005; Freund's complete adjuvant (for animals); Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300 adjuvant; alum; QS 21, muramyl dipeptide; trehalose; bacterial extracts, including mycobacterial extracts; detoxified endotoxins; membrane lipids; or combinations thereof.

[0131] Monoclonal antibodies can be produced using hybridoma technology in accordance with methods well known to those skilled in the art. The antibodies are useful as research or diagnostic reagents or can be used for passive immunization. The composition may optionally contain an adjuvant.

[0132] The polyclonal and monoclonal antibodies useful as research or diagnostic reagents may be employed for detection and measurement of SEQ ID NO:2, SEQ ID NO:4, and fragments or conservative substitutions thereof. Such antibodies may be used to detect these proteins in a biological sample, including but not limited to samples such as cells, cellular extracts, tissues, tissue extracts, biopsies, tumors, and biological fluids. Such detection capability is useful for detection of disease related to these proteins to facilitate diagnosis and prognosis and to suggest possible treatment alternatives.

[0133] Detection may be achieved through the use of immunocytochemistry, ELISA, radioimmunoassay or other assays as commonly known to one of ordinary skill in the art. The Nox 4 and Nox 5 proteins, or fragments or conservative substitutions thereof, may be labeled through commonly known approaches, including but not limited to the following: radiolabeling, dyes, magnetic particles, biotin-avidin, fluorescent molecules, chemiluminescent molecules and systems, ferritin, colloidal gold, and other methods known to one of skill in the art of labeling proteins.

[0134] Administration of Antibodies

[0135] The antibodies directed to the proteins shown as SEQ ID NO:2, SEQ ID NO:4, or directed to fragments or conservative substitutions thereof, may also be administered directly to humans and animals in a passive immunization paradigm. Antibodies directed to extracellular portions of SEQ ID NO:2, SEQ ID NO:4, or fragments thereof bind to these extracellular epitopes. Attachment of labels to these antibodies facilitates localization and visualization of sites of binding. Attachment of molecules such as ricin or other cytotoxins to these antibodies helps to selectively damage or kill cells expressing SEQ ID NO:2, SEQ ID NO:4, or fragments thereof.

[0136] Kits

[0137] The present invention includes kits useful with the antibodies, nucleic acid probes, labeled antibodies, labeled proteins or fragments thereof for detection, localization and measurement of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or combinations thereof and fragments or conservative substitutions thereof. The diagnostic kits may also measure or detect the relative expression of the Nox proteins described herein (i.e. human Nox 4 and/or human Nox 5)

[0138] Kits may be used for immunocytochemistry, in situ hybridization, solution hybridization, radioimmunoassay, ELISA, Western blots, quantitative PCR, and other assays for the detection, localization and measurement of these nucleic acids, proteins or fragments thereof using techniques known to one of skill in the art.

[0139] The nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or fragments thereof, may also be used under high stringency conditions to detect alternately spliced messages related to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or fragments thereof, respectively.

[0140] Fragments of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, containing the relevant hybridizing sequence can be synthesized onto the surface of a chip array. RNA samples, e.g., from tumors, are then fluorescently tagged and hybridized onto the chip for detection. This approach may be used diagnostically to characterize tumor types and to tailor treatments and/or provide prognostic information. Such prognostic information may have predictive value concerning disease progression and life span, and may also affect choice of therapy.

[0141] The other present invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention.

EXAMPLE 1

[0142] Sequence Analysis and Cloning of the Human Nox 4 cDNA (SEQ ID NO:1) Encoding for Production of the Human Nox 4 Protein (SEQ ID NO:2)

[0143] Using Nox 3 (SEQ ID NO:6) as a query sequence, a 789 base pair sequenced portion of expressed sequence tag (EST) GenBank Accession number AI742260) and a 408 base EST clone (GenBank No. A1885681, a clone exhibiting a 26% identity to the cDNA sequence corresponding to amino acid residues 433-560 of Nox 3, and a second clone showing 36% identity to the cDNA sequence corresponding to amino acid residues 5-48 of Nox 3 were discovered. This homologue was cloned using two PCR primers based on the two EST sequences: (SEQ ID NO:7, 5′-CAACGAAGGGGTTAAACACCTCTGC-3′; and SEQ ID NO:8, 5′-CACAGCTGATTGATTCCGCTGAG-3′). PCR was carried out using human fetal kidney marathon-ready cDNA (Clontech, Palo Alto, Calif.), and the 0.85 kb product was sequenced. Based on the sequencing results, 5′- and 3′-RACE using the same library using the following primers: 5′-RACE: SEQ ID NO:9, 5′-TAAGCCAAGAGTGTTCGGCACATG-3′; SEQ ID NO:10, 5′-TACTCTGGCCCTTGGTTATACAGCA-3′(for nested PCR); 3′-RACE: SEQ ID NO:11, 5′-TCCATTTACCCTCACAATGTGT-3′; SEQ ID NO:12, 5′-CTCAGCGGAATCAATCAGCTGTG-3′(for nested PCR) was then carried out. PCR parameters were 95° C. for 30 s, 62° C. or 65° C. for 20 s, 72° C. for 45 s, 25-35 cycles as indicated after denaturing for 1 min 30 s at 95° C. PCR products were purified with a QIAquick PCR purification kit or a gel purification kit (QIAGEN, Valencia, Calif.). The positive PCR bands were sequenced by ABI 377 automatic sequencing. Primers were designed to subclone the full-length cDNA and the correct sequence was confirmed by automated sequencing.

[0144] Secretion signal sequences were predicted according to web-based SMART program (version 3.1) at EMBL (http://www.smart.embl-heidelberg.de/smart/). Prediction of open reading frames (ORF) was carried out using the EditSeq program (DNASTAR), and phylogenetic analyses and multiple sequence alignment were carried out using the clustal method using the Megalign program (DNASTAR). Transmembrane alpha helices were predicted using the TMHMM algorithms through the Center for Biological Sequence Analysis (http://genome.cbs.dtu.dk/services/TMHMM/).

[0145] Total RNA was extracted from cell lines with Trizol (Life Technologies, Gaithersburg, Md.) based on the manufacturer's protocol or according to (Ishii et al., 1999) for glioma cell lines. RNAs were reverse transcribed into first-strand cDNA with Superscript II (Life Technologies, Gaithersburg, Md.) using oligo-dT according on the method provided by the manufacturer.

[0146] Table 4 shows the basic features of the cDNA and the predicted proteins. Like the proteins encoded by gp91phox (a.k.a., Nox 2 SEQ ID NO:13) and Nox 1 (SEQ ID NO:14), the new sequences encode predicted proteins of around 65 kDa, and message sizes are similar in length (2.0-2.2 kb). Nox 4 also has 59 amino acids which are strongly basic, 45 amino acids which are strongly acidic, 212 hydrophobic amino acids, 171 polar amino acids, an isoelectic point of 8.695 and a charge of 16.549 at a pH of 7.0. Nox 4 also shows 21-59% identity with gp91phox and with Nox 1. Nox 1, gp91phox, Nox 3 and Nox 4 cluster within a sub-family that is similar to gp91 phox. The alignment of the predicted protein sequence of Nox 4 is shown in FIG. 2. The molecules are roughly divided into two large domains: an N-terminal cluster of hydrophobic membrane-spanning sequences, and a C-terminal flavoprotein domain. The latter shows weak homology with a number of FAD binding proteins including cytochrome P-450 reductase and ferredoxin-NADP oxidoreductase (Rotrosen et al., 1992; Segal et al., 1992). Within the flavoprotein domain are two segments (indicated in FIGS. 2(a-b)) that show homology with known FAD binding sites in other flavoproteins, and four segments closer to the C-terminus that are homologous to documented pyridine nucleotide binding sites in other proteins. The first of these includes the G-X-G-X-X-P canonical sequence that characterizes pyridine nucleotide binding sites. In all Nox forms, this sequence is followed by an F, which is typical of NADPH- rather than NADH-specific enzymes.

[0147] Nox 4 contains the predicted transmembrane alpha helix near the extreme N-terminus (light hashed box in FIG. 2). However, this region is also strongly predicted to be a signal peptide sequence in these forms. Predicted proteolytic cleavage sites for each isoform are indicated by the arrows, and cleavage at these positions would lead to a loss of the first putative transmembrane sequence. Five additional transmembrane regions are also predicted in this protein. The most C-terminal of these is weakly predicted in Nox 1, gp91phox, Nox 3 and Nox 4 and is entirely missed by some prediction algorithms. It is necessary to include this transmembrane region in order to generate a model (FIG. 3) which is consistent with known features of gp91phox, particularly a cytosolic facing location of the flavoprotein domain. In this model, known N-linked glycosylation sites in gp91phox are correctly localized to extracellular loops (although these sites are not conserved in other isoforms). In addition, a polybasic loop of gp91phox that binds to the cytosolic regulatory protein p47phox (Biberstine-Kinkade et al., 1999) is localized on the cytosolic face. In general, extracellular loops tend to be highly variable in length and sequence, whereas the transmembrane helices and intracellular loops tend to be more conserved in sequence and length (FIG. 2).

[0148] Within the N-terminus are five absolutely conserved histidines (FIG. 2), that are also conserved in all other members of the Nox family of enzymes. gp91phox contains two heme groups, the irons of which are each ligated by two histidyl nitrogens (Isogai et al., 1993), and these are thought to reside within the N-terminus (Yu et al., 1998). An additional conserved histidine lies within the FAD-binding region and is therefore not a candidate for heme ligation. Thus, four of the five histidines within the N-terminus probably participate in heme ligation, providing part of the binding sites for two heme groups, as indicated in FIG. 3. TABLE 4 Molecular Features of Nox 3, Nox 4, and Nox 5 cDNA Nox 3 Nox 4 Nox 5 cDNA length (bp) 2044 2232 2199 Predicted number of 568 578 565 amino acids Predicted protein Mw 64.9 66.9 64.7 (kDa) pI of protein 8 8.7 9.7 Kozek sequence ATCATGA or GGCATGG GTCATGG ATGATGG Identity to gp91phox 58% 37% 27% Identity to Nox I 55% 35% 29% GenBank accession No. AF190122 AF254621 AF317889

EXAMPLE 2

[0149] Tissue Expression of Nox 4 mRNA

[0150] The predominant Nox 4 2.4 kb message, which corresponds to the size expected for the full-length Nox 4 transcript, is highly expressed in adult as well as fetal kidney (FIG. 4A). An additional weak Nox 4 band was also detected at 4.5 kilobases (kb) in fetal and adult kidney (FIG. 4A). It is particularly expressed at the site of erythropoietin production in the kidney. RT-PCR confirmed kidney expression and also revealed expression of Nox 4 in all fetal tissues tested as well as in several adult tissues including pancreas, placenta, ovary, testis and skeletal muscle.

EXAMPLE 3

[0151] Sequence Analysis and Cloning of the cDNA for Human Nox 5

[0152] The Blast search using Nox 3 (SEQ ID NO:6) as a query sequence also identified homology with two unfinished genomic clones, GenBank No. AC027088 and AC026512, respectively. These clones exhibit 46 to 50% identity to Nox 3 within three exons. 5′- and 3′- RACE were carried out using human fetal kidney marathon-ready cDNA (Clontech, Palo Alto, Calif.), using the following four primers which were designed based on the genomic sequence: SEQ ID NO:15, 5′-CTCATTGTCACACTCCTCGACAGC-3′; SEQ ID NO:16, 5′-TGGGTCTGATGCCTTGAAGGACTC-3′(for nested PCR); 3′-RACE: SEQ ID NO:17, 5′-ATCAAGCGGCCCCCTTTTTTTCAC-3′; SEQ ID NO:18, 5′-CTGAACATCCCCACCATTGCTCGC-3′(for nested PCR). PCR parameters were 95° C. for 30 s, 62° C. or 65° C. for 20 s, 72° C. for 45 s, 25-35 cycles as indicated after denaturing for 1.5 minutes at 95° C. PCR products were purified with a QIAquick PCR purification kit or a gel purification kit (QIAGEN, Valencia, Calif.). Primers were designed to subclone the full-length cDNA and the correct sequence was confirmed by ABI 3777 automated sequencing.

[0153] Secretion signal sequences were predicted according to web-based SMART program (version 3.1) at EMBL (http://www.smart.embl-heidelberg.de/smart/). Prediction of open reading frames (ORF) was carried out using the EditSeq program (DNASTAR), and phylogenetic analyses and multiple sequence alignment were carried out using the clustal method using the Megalign program (DNASTAR). Transmembrane alpha helices were predicted using the TMHMM algorithms through the Center for Biological Sequence Analysis (http://genome.cbs.dtu.dk/services/TMHMM/).

[0154] Total RNA was extracted from cell lines with Trizol (Life Technologies, Gaithersburg, Md.) based on the manufacturer's protocol or according to Ishii et al., (1999) for glioma cell lines. RNAs were reverse transcribed into first-strand cDNA with Superscript II (Life Technologies, Gaithersburg, Md.) using oligo-dT according on the method provided by the manufacturer.

[0155] Table 4 shows the basic features of the cDNA and the predicted proteins. Like the proteins encoded by gp91phox (SEQ ID NO:13) and Nox 1 (SEQ ID NO:14), the new sequences for Nox 4 and Nox 5 encode predicted proteins of around 65 kDa, and message sizes are similar in length (2.0-2.2 kb). Nox 3, Nox 4 and Nox 5 show 21-59% identity with gp91phox. Nox 5 forms a unique group, of which it is the only member identified to date, and which is highly divergent from other members of the family. Based on its position in the family tree, Nox 5 may represent the gene which is closest to the primordial Nox.

[0156] The alignment of the predicted protein sequences of gp91phox, Nox 1, Nox 3, Nox 4 and Nox 5 is shown in FIG. 2. The molecules are roughly divided into two large domains: an N-terminal cluster of hydrophobic membrane-spanning sequences, and a C-terminal flavoprotein domain. The latter shows weak homology with a number of FAD binding proteins including cytochrome P-450 reductase and ferredoxin-NADP oxidoreductase (Rotrosen et al., 1992; Segal et al., 1992). Within the flavoprotein domain are two segments (indicated in FIG. 2) that show homology with known FAD binding sites in other flavoproteins, and four segments nearer the C-terminus that are homologous to documented pyridine nucleotide binding sites in other proteins.

[0157] The first of these includes the G-X-G-X-X-P canonical sequence that characterizes pyridine nucleotide binding sites. In all Nox forms, this sequence is followed by an F, which is typical of NADPH- rather than NADH-specific enzymes.

[0158] While the N-terminal half of Nox 1, Nox 3, Nox 4, and Nox 5 are all hydrophobic, Nox 5 differs from the others somewhat in the details of predicted transmembrane alpha helices, as illustrated in FIG. 2. Nox 5 does not contain an N-terminal predicted signal peptide, but does contain a predicted transmembrane alpha helix (first hashed box, Nox 5 sequence in FIG. 2). According to the prediction algorithm, the extreme N-terminus of Nox 5 is located on the inside of the membrane, on the same side as the flavoprotein domain. Five additional transmembrane regions are also predicted in these proteins. The most C-terminal of these is strongly predicted in Nox 5. It is necessary to include this transmembrane region in order to generate a model (FIG. 3) which is consistent with known features of gp91phox, particularly a cytosolic facing location of the flavoprotein domain. In this model, known N-linked glycosylation sites in gp91phox are correctly localized to extracellular loops (although these sites are not conserved in other isoforms). In addition, a polybasic loop of gp91phox that binds to the cytosolic regulatory protein p47phox (Biberstine-Kinkade et al., 1999) is localized on the cytosolic face. In general, extracellular loops tend to be highly variable in length and sequence, whereas the transmembrane helices and intracellular loops tend to be more conserved in sequence and length (FIG. 2).

[0159] Within the N-terminus are five absolutely conserved histidines (FIG. 2), that are also conserved in all other members of the Nox family of enzymes (data not shown). gp91phox contains two heme groups, the irons of which are each ligated by two histidyl nitrogens (Isogai et al., 1993), and these are thought to reside within the N-terminus (Yu et al., 1998). An additional conserved histidine lies within the FAD-binding region and is therefore not a candidate for heme ligation. Thus, four of the five histidines within the N-terminus probably participate in heme ligation, providing part of the binding sites for two heme groups, as indicated in FIG. 3.

[0160] Additionally, located at the extreme N-terminus on the cytosolic side of the membrane of Nox 5 is a highly cationic proline-rich sequence (the Pro-Arg-Rich sequence indicated in FIG. 2 and FIG. 3). This region is thought to serve as a binding sequence for Src-Homology 3 (SH3) domains in another protein. SH3 domains are known to recognize inter- or intra-molecular proline-rich sequences. This is similar to p22phox, a membrane-associated subunit that associates with gp91phox, and contains a C-terminal, proline-rich sequence (Parkos et al., 1988) that serves as a binding site for a SH3 domain in p47phox, one of the cytosolic subunits that regulates the activity of gp91phox. Although not wanting to be bound by the following statement, it is possible that the proline-rich sequence in Nox 5 serves as an internal p22phox, allowing interaction with cytosolic regulatory proteins.

EXAMPLE 4

[0161] Tissue Expression of Nox 5 mRNA

[0162] Northern blots probed for Nox 5 using a 3′-portion of the coding region (FIG. 4A) revealed the presence of a 2.2 kb band corresponding in size to the full-length Nox 5 transcript in all fetal tissues tested. This species was also seen in low amounts in adult spleen and testis, along with larger transcripts at 2.6 kb and 6 kb. A probe using a portion of the 3′ untranslated region also revealed the presence of the same 2.6 kb and 6 kb bands (FIG. 4B). Thus, these larger bands are larger transcripts derived from the same gene. RT-PCR confirmed expression of Nox 5 in testis and spleen, and also revealed weak expression in ovary, placenta, and pancreas (FIG. 5).

EXAMPLE 5

[0163] Real Time RT-PCR of Nox 4 and Nox 5.

[0164] G3PDH was used as a control. The G3PDH PCR product was purified using a QIAquick PCR purification kit (QIAGEN, Valencia, Calif.) and quantified using absorbance at 260 nm using a BECKMAN DU640B spectrophotometer. The standard curve for G3PDH was constructed using 10-fold serial dilutions of a known concentration of G3PDH PCR product in distilled water. Real time PCR amplification was carried out using a LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) in a PCR reaction containing 0.2 μM of each primer, 1: 84,000 SYBR Green I (Molecular Probes, Eugene, Oreg.) and Advantage 2 Polymerase Mix (Clontech, Polo Alto, Calif.). Amplification was carried out for 36 cycles of denaturation (95° C., 0 s, ramp rate 20°/s), annealing (65° C., 5 s, ramp rate 20/s) and extension (72° C., 30 s ramp rate 20° C./s). Fluorescence was monitored at the end of each extension phase. Quantitation and melting curve were analyzed with the LightCycler software. RT-PCR confirmed kidney expression and also revealed expression of Nox 4 in all fetal tissues tested as well as in several adult tissues including pancreas, placenta, ovary, testis and skeletal muscle. (See FIG. 5) The ratio of copies of unknown to standard G3PDH was then calculated and is reported in FIG. 6C. RT-PCR also confirmed expression of Nox 5 in testis and spleen, and revealed weak expression in ovary, placenta, and pancreas (FIG. 5). The data indicate that expression patterns of Nox family members are tissue specific, and do not correspond to the expression of gp91 phox.

EXAMPLE 6

[0165] Northern Blotting of Nox 4 and Nox 5

[0166] The Human Fetal and Adult Multiple Tissue Northern Blot (Clontech, Palo Alto, Calif.) was hybridized with ³²P random primer-labeled Nox 4, or Nox probe according to the manufacturer's instructions. The probes were prepared by PCR with primers for Nox 4: SEQ ID NO:7 and SEQ ID NO:8; and primers for Nox 5: SEQ ID NO:19, 5′-CTGAACATCCCCACCATTGCTCGC-3′ and SEQ ID NO: 20, 5′-GAAGCCGAACTTCTCACAATGGCC-3′. The PCR products represent coding sequences corresponding to amino acids 11-294 (Nox 4), or 278-557 (Nox 5). Because the Nox 5 transcript sizes differ between fetal and adult northern blots, a 420bp PCR product of the Nox 5 3′-untranslated region amplified by primers (SEQ ID NO: 21 5′-CCTCACCTCTCCAAGCTCTGCCCC-3′ and SEQ ID NO: 22 5′-TTGAACAATTTTATAAGATGCCGG-3′) was also used to hybridize Northern Blots.

[0167] The predominant Nox 4 2.4 kb message, which corresponds to the size expected for the full-length Nox 4 transcript, is highly expressed in adult as well as fetal kidney (FIG. 4A), confirming recent reports (Kikuchi et al., 2000; Geiszt et al., 2000; Shiose et al., 2000). An additional weak Nox 4 band was also detected at 4.5 kilobases (kb) in fetal and adult kidney (FIG. 4A). Northern blots probed for Nox 5 (FIG. 4A) revealed the presence of a 2.2 kb band corresponding in size to the full-length Nox 5 transcript in all fetal tissues tested. This species was also seen in low amounts in adult spleen and testis, along with larger transcripts at 2.6 kb and 6 kb. A probe using a portion of the 3′ untranslated region also revealed the presence of the same 2.6 kb and 6 kb bands (FIG. 4B). Thus, these larger bands are larger transcripts derived from the same gene.

EXAMPLE 7

[0168] Transfection of NIH3T3 Cells with SEQ ID NO:1 or SEQ ID NO:3

[0169] The nucleotide sequence SEQ ID NO:1 or SEQ ID NO:3 encoding for production of the Nox 4 protein (SEQ ID NO:2) or the Nox 5 protein (SEQ ID NO:4), respectively, is subcloned into the Not1 site of the pEF-PAC vector (obtained from Mary Dinauer, Indiana University Medical School, Indianapolis, Ind.) which has a puromycin resistance gene. Transfection is carried out as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Volumes 1-3, 2nd edition, Cold Spring Harbor Laboratory Press, N.Y., 1989. The SEQ ID NO:1 in pEF-PAC and the empty vector are separately transfected into NIH 3T3 cells using Fugene 6 (Boeringer Mannheim).

[0170] 10⁵ to 10³ cells stably transfected separately with human Nox 4 gene SEQ ID NO:1, with human Nox 5 gene SEQ ID NO:3, and with empty vector are prepared in 0.3% warm (40° C.) agar solution containing DMEM and 10% calf serum. Cells are distributed onto a hardened 0.6% agar plate prepared with DMEM and 10% calf serum. After three weeks in culture (37° C., 5% CO₂) colony formation is observed by microscopy.

[0171] About 2×10⁶ cells maintained in DMEM containing 10% calf serum are transfected with 10 μg of DNA. After 2 days, cells are split and selected in the same medium containing 1 mg/ml puromycin. Colonies that survive in selection media for 10 to 14 days are subcultured continuously in the presence of puromycin.

[0172] Cells which are stably transfected with the empty vector and cultured in soft agar for 3 weeks as above do not display anchorage independent growth. In contrast, NIH 3T3 cells which are stably transfected with the Nox 4 (SEQ ID NO:1) or with the Nox 5 gene (SEQ ID NO:3) cultured for 3 weeks in soft agar demonstrate anchorage independent growth of colonies. Transfected cells exhibit a transformed-like morphology, similar to that seen with (V 12) Ras-transfected cells, characterized by long spindle-like cells.

EXAMPLE 8

[0173] Expression of Nox 4 (SEQ ID NO.1) or Nox 5 (SEQ ID NO:3) in Transfected NIH3T3 Cells

[0174] To verify the expression of Nox 4 mRNA or Nox 5 mRNA after transfection, RT-PCR and Northern blotting are performed. Total RNAs are prepared from 10⁶ cells using the High Pure RNA Isolation Kit (Boeringer Mannheim) or Rneasy kit (Qiagen). cDNAs for each colony are prepared from 1-2 μg of total RNA using Advantage RT-PCR Kit (ClonTech). PCR amplification is performed using primers, SEQ ID NO: 23 and SEQ ID NO:24. For Northern blotting, 10-20 μg of total RNA is separated on a 1% agarose formaldehyde gel and transferred to a nylon filter. After ultraviolet (UV) cross-linking, filters are used for Northern blotting assay as described in Example 6. Colonies expressing large amounts of Nox 4 mRNA or Nox 5 mRNA are chosen for further analysis.

EXAMPLE 9

[0175] NADPH-Dependent Superoxide Generation Assay

[0176] In one embodiment of the present invention, NIH 3T3 cells stably transfected with the human Nox 4 gene (SEQ ID NO:1) or human Nox 5 gene (SEQ ID NO:3) are analyzed for superoxide generation using the lucigenin (Bis-N-methylacridinium luminescence assay (Sigma, St. Louis, Mo., Li et al. (1998) J. Biol. Chem. 273, 2015-2023). Cells are washed with cold HANKS' solution and homogenized on ice in HANKS' buffer containing 15% sucrose using a Dounce homogenizer. Cell lysates are frozen immediately in a dry ice/ethanol bath. For the assay, 30 μg of cell lysate is mixed with 200 μM NADPH and 500 μM lucigenin. Luminescence is monitored using a LumiCounter (Packard) at three successive one minute intervals and the highest value was used for comparison. Protein concentration is determined by the Bradford method.

[0177] Superoxide generation is monitored in lysates from some of the stably transfected cell lines and is compared with superoxide generation by the untransfected NIH 3T3 cell lysates. The luminescent signal is inhibited by superoxide dismutase and the general flavoprotein inhibitor diphenylene iodonium, but is unaffected by added recombinant human p47phox, p67phox and Rac1(GTP-γS), which are essential cytosolic factors for the phagocyte respiratory-burst oxidase.

[0178] In an alternate and preferred embodiment of the present invention, cells that are stably transfected with Nox 4 (YA28), Nox 5 (YA28) or with empty vector (NEF2) are grown in 10 cm tissue culture plates in medium containing DMEM, 10% calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml puromycin to approximately 80% confluency. Cells (five tissue culture plates of each cell type) are washed briefly with 5 ml phosphate buffered saline (PBS) then dissociated from the plates with PBS containing 5 mM EDTA. Cells are pelleted by centrifuging briefly at 1000× g.

[0179] To permeabilize the cells, freeze thaw lysis is carried out and this is followed by passage of the cell material through a small bore needle. The supernatant is removed and the cells are frozen on dry ice for 15 minutes. After cells are thawed, 200 μl lysis buffer (HANKS' Buffered Salt Solution-HBBS) containing a mixture of protease inhibitors from Sigma (Catalog no.

[0180] P2714) is added. Cells on ice are passed through an 18 gauge needle 10 times and 200 μl of HBSS buffer containing 34% sucrose was added to yield a final concentration of 17% sucrose. Sucrose appeared to enhance stability upon storage. The combination of freeze-thawing and passage through a needle results in lysis of essentially all of the cells, and this material is referred to as the cell lysate.

[0181] The cell lysates are assayed for protein concentration using the BioRad protein assay system. Cell lysates are assayed for NADPH-dependent chemiluminescence by combining HBSS buffer, arachidonic acid, and 0.01-1 μg protein in assay plates (96 well plastic plates). The reaction is initiated by adding 1.5 mM NADPH and 75 μM lucigenin to the assay mix to give a final concentration of 200 μM NADPH and 10 μM lucigenin, and the chemiluminescence is monitored immediately. The final assay volume as 150 μl. The optimal arachidonic acid concentration is between 50-100 μM. A Packard Lumicount luminometer is used to measure chemiluminescence of the reaction between lucigenin and superoxide at 37° C. The plate is monitored continuously for 60 minutes and the maximal relative luminescence unit (RLU) value for each sample is used for the graph.

[0182] The presence of NaCl or KCl within a concentration range of 50-150 μM is important for optimal activity. MgCl₂ (1-5 mM) further enhanced activity by about 2-fold. This cell-free assay for Nox 4 NADPH-oxidase activity and the cell-free assay for Nox 5 NADPH-oxidase are useful for screening modulators (inhibitors or stimulators) of the Nox 4 enzyme and Nox enzyme. The assay may also be used to detect Nox NADPH-oxidase activity in general and to screen for modulators (inhibitors or stimulators) of the Nox family of enzymes.

EXAMPLE 10

[0183] Nitro Blue Tetrazolium Reduction by Superoxide Generated by NIH 3T3 Cells Transfected with the Nox 4 cDNA (SEQ ID NO:1) or the Nox 5 cDNA (SEQ ID NO:3)

[0184] Superoxide generation by intact cells is monitored by using superoxide dismutase-sensitive reduction of nitroblue tetrazolium. NEF2 (vector alone control), YA26 (Nox 4 (SEQ ID NO:1)-transfected), YA26 (Nox 5 (SEQ ID NO:3)-transfected), YA28 (Nox 4 (SEQ ID NO:1)-transfected) and YA28 (Nox 5 (SEQ ID NO:3)-transfected) cells are plated in six well plates at 500,000 cells per well. About 24 hours later, medium is removed from cells and the cells are washed once with 1 mL Hanks solution (Sigma, St. Louis, Mo.). About 1 mL of filtered 0.25% Nitro blue tetrazolium (NBT, Sigma) is added in Hanks without or with 600 units of superoxide dismutase (Sigma) and cells are incubated at 37° C. in the presence of 5% CO₂. After 8 minutes the cells are scraped and pelleted at more than 10,000 g. The pellet is re-suspended in 1 mL of pyridine (Sigma) and heated for 10 minutes at 100° C. to solubilize the reduced NBT. The concentration of reduced NBT is determined by measuring the absorbance at 510 nm, using an extinction coefficient of 11,000 M⁻¹cm⁻¹. Some wells are untreated and used to determine cell number. Because superoxide dismutase is not likely to penetrate cells, superoxide must be generated extracellularly. The amount of superoxide generated by these cells is about 5-10% of that generated by activated human neutrophils.

EXAMPLE 11

[0185] Modification of Intracellular Components in Nox 4 and Nox 5 Transfected Cells

[0186] To test whether superoxide generated by Nox 4 or Nox 5 can affect intracellular targets, aconitase activity in control and Nox-transfected cell lines is monitored using a method as described in Suh et al. (1999) Nature 401, 79-82. Aconitase contains a four-iron-sulphur cluster that is highly susceptible to modification by superoxide, resulting in a loss of activity, and has been used as a reporter of intra-cellular superoxide generation. Acotinase activity is determined as described in Gardner et al. (1995) J. Biol. Chem. 270, 13399-13405. Acotinase activity is significantly diminished in the Nox-transfected cell lines designated YA26, and YA28 as compared to the transfected control.

[0187] Approximately 50% of the aconitase in these cells is mitochondrial, based on differential centrifugation, and the cytosolic and mitochondrial forms were both affected. Control cytosolic and mitochondrial enzymes that do not contain iron-sulfur centers are not affected. Superoxide generated in Nox 4-transfected cells and Nox 5-transfected cells is therefore capable of reacting with and modifying intracellular components.

EXAMPLE 12

[0188] Tumor Generation in Nude Mice Receiving Cells Transfected with the Human Nox 4 cDNA (SEQ ID NO:1) or the Human Nox 5 cDNA (SEQ ID NO:3)

[0189] About 2×10⁶ NIH 3T3 cells (either Nox 4-transfected with SEQ ID NO:1, Nox 5-transfected with SEQ ID NO:3, or cells transfected using empty vector) are injected subdermally into the lateral aspect of the neck of 4-5 week old nude mice. Three to six mice are injected for each of three Nox 4-transfected cell lines, each of the Nox 5-transfected cell lines, and 3 mice are injected with the cells transfected with empty vector (control). After 2 to 3 weeks, mice are sacrificed. The tumors are fixed in 10% formalin and characterized by histological analysis.

[0190] In another study, 15 mice are injected with Nox 4-transfected NIH 3T3 cells. Of the 15 mice injected, 14 show large tumors within 17 days of injection, and tumors show expression of Nox 4 mRNA.

[0191] In another study, 15 mice are injected with Nox 5-transfected NIH 3T3 cells. Of the 15 mice injected, 14 show large tumors within 17 days of injection, and tumors show expression of Nox 5 mRNA.

EXAMPLE 13

[0192] Demonstration of the Role of Nox 4 and Nox 5 in Non-Cancerous Growth

[0193] A role for Nox 4 and Nox 5 in normal growth is demonstrated in rat aortic vascular smooth-muscle cells by using antisense to Nox 4 or Nox 5. Transfection with the antisense DNA results in a decrease in both superoxide generation and serum-dependent growth. Nox 4 and Nox 5 are therefore implicated in normal growth in this cell type.

EXAMPLE 14

[0194] Expression of Human Nox 4 Protein (SEQ ID NO:2) and Human Nox 5 Protein (SEQ ID NO:4) in a Baculovirus Expression System

[0195] SEQ ID NO:2 and SEQ ID NO:4 are also expressed in insect cells using recombinant baculovirus. To establish the Nox 4 and Nox 5 expressing virus systems, the Nox 4 gene (SEQ ID NO:1) or the Nox 5 gene (SEQ ID NO:5) is initially cloned separately into the pBacPAK8 vector (Clontech, Palo Alto, Calif.) and recombinant baculovirus is constructed using standard methods according to manufacturer's protocols. Briefly, PCR amplified Nox 4 DNA or Nox 5 DNA is cloned into the KpnI and EcoRI site of the vector. Primers used for PCR amplification are SEQ ID NOs:21, 22, 23 and 24. Sf9 insect cells (2×10⁶ cells) are infected with 0.5 mg of linearized baculovirus DNA sold under the trademark BACULOGOLD® (PharMingen, San Diego, Calif.) and 5 mg pBacPAC8 Nox 4 using Transfection Buffers A and B (PharMingen, San Diego, Calif.). After 5 days, the supernatants containing recombinant viruses are harvested and amplified by infecting fresh sf9 cells for 7 days. Amplification is carried out three times and the presence of the recombinant viruses containing Nox 4 DNA or Nox 5 DNA is confirmed by PCR using the same primers. After three times amplification of viruses, plaque purification are carried out to obtain the high titer viruses. Approximately 2×10⁸ sf9 cells in agar plates are infected for 5 days with serial dilutions of virus and are dyed with neutral red for easy detection of virus plaques. Selected virus plaques are extracted and the presence of the human Nox 4 DNA or human Nox 5 DNA is confirmed again by PCR.

EXAMPLE 15

[0196] Antibodies to Human Nox 4 (SEQ ID NO:2) and Human Nox 5 (SEQ ID NO:4)

[0197] Polyclonal antibodies are raised separately in rabbits against human Nox 4 (SEQ ID NO:2) or human Nox 5 (SEQ ID NO: 4). Proteins are separately conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehyde.

[0198] Antigens are injected into different rabbits initially in complete Freund's adjuvant, and are boosted 4 times with antigen in incomplete Freund's adjuvant at intervals of every three weeks. Approximately 0.5 mg to 1 mg of peptide is administered at each injection. Blood is drawn 1 week after each boost and a terminal bleed is carried out 2 weeks after the final boost. Anti Nox 4 and anti Nox 5 antibodies are purified on affinity columns to which are bound Nox 4 or Nox 5 using techniques known to one of ordinary skill in the art. Unbound protein is washed away with 20 ml of buffer. Elution of the antibodies from the gel was performed with 6 ml of elution buffer (100 mM glycine/HCl, pH 2.5, 200 mM NaCl, and 0.5% Triton X-100). The eluate is then neutralized by adding 0.9 ml of 1 M Tris/HCl, pH 8.0.

[0199] The detection of antigens is performed using an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK). The affinity purified antibodies to Nox 4 or to Nox 5 are used at a dilution of 1:1000 in a Western blot in which a total of 10 μg of protein is added to each lane.

EXAMPLE 16

[0200] Construction of a Reporter Construct for Nox-1

[0201] pGL3-basic (Promega, Madison, Wis.) was used as the parent vector.

[0202] The pGL3-basic vector lacks eukaryotic promoter and enhancer sequences, allowing for maximum flexibility in cloning putative regulatory sequences. Expression of luciferase activity in cells transfected with this plasmid depends on insertion and proper orientation of a functional promoter upstream from luc+. Potential enhancer elements can also be inserted upstream of the promoter or in the BamH I or Sal I sites downstream of the luc+gene. Primers SEQ ID NO: 25, 5′GCTACTCGAGTGTGCCAATTTCACCTGGCAT-3′ and SEQ ID NO:26, 5 ′-AACTCTCGAGTGTCAAGAGGTGGTTTGGAGC-3′ were used along with genomic DNA to obtain the promoter region of Nox 1 (SEQ ID NO:5) flanked by Xho restriction sites. The restriction sites were then used to insert the Nox 1 promoter region into the pGL3 plasmid. (See FIG. 7). Successful transfection was determined by the activity of luciferase which was measured using a luminometer.

EXAMPLE 17

[0203] Use of the Reporter Construct as an Assay

[0204] The construct from Example 16 is stably transfected into human Caco-2 or HT-29 cells. Transfection is carried out as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Volumes 1-3, 2nd edition, Cold Spring Harbor Laboratory Press, N.Y., 1989. The SEQ ID NO:5 in pEF-PAC and the empty vector are separately transfected into Caco-2 cells using Fugene 6 (Boeringer Mannheim). 10⁵ to 10³ cells stably transfected with human Nox 1 promoter gene SEQ ID NO:5 and with empty vector are prepared in 0.3% warm (40° C.) agar solution containing DMEM and 10% calf serum. Cells are distributed onto a hardened 0.6% agar plate prepared with DMEM and 10% calf serum. After three weeks in culture (370C, 5% CO₂) colony formation is observed by microscopy. About 2×10⁶ cells maintained in DMEM containing 10% calf serum are transfected with 10 μg of DNA. After 2 days, cells are split and selected in the same medium containing 1 mg/ml puromycin. Colonies that survive in selection media for 10 to 14 days are subcultured continuously in the presence of puromycin.

[0205] The colonies are used as a screening assay by adding compounds to the media suspected of effecting the expression of ROI. Measurement of the luciferase output indicates whether a compound enhanced or inhibited the induction of the Nox 1 gene and facilitates the development of drugs based on a compound's cellular effects.

EXAMPLE 18

[0206] Expression of Nox 3, Nox 4, and Nox 5 mRNA in Cancer Cells

[0207] Many cancer cells overproduce reactive oxygen species (Szatrowski and Nathan, 1991), and this may be causative in the transformed phenotype (Suh et al., 1999). The expression of Nox 1-5 was investigated in a variety of human tumor and other cell lines, to determine if these enzymes might account for reactive oxygen generation seen in some tumors. Expression of Nox family members in human cancers. RT-PCR was carried out as in FIG. 5. FIG. 6A shows Nox expression in the following cell lines: ES-2 (ovarian clear cell carcinoma), PA-1 (ovarian teratocarcinoma), Ovcar-3 (ovarian adenocarcinoma), MDA-MB-231 (mammary adenocarcinoma), SKO-007 (plasmacytoma), Caco-2 (colon carcinoma), T84 (colon carcinoma), HEK293 (embryonic kidney transformed with adenovirus), and Hela (cervical adenocarcinoma). FIG. 6B show Nox expression in five cell lines derived from human glioblastomas, as well as from human astrocyte primary cultures. FIG. 6C shows the ratio of expression of gp91phox, Nox 4 and Nox 5 compared with G3PDH, obtained from real time PCR results.

[0208] As shown in FIGS. 6A and 6B, Nox isoforms were expressed in 12 out of the 14 tumor or transformed cell lines examined. Nox 1 is expressed in two colon cancer lines, Caco-2 and T-84, as well as in the transformed cell line HEK293, and to a lesser extent in Hela cells. Nox 4 was seen in 11 of these cell lines, while Nox 5 was seen in 7. gp91phox was also expressed in more than half of the cell lines. The identity of the mRNAs was confirmed by sequencing as indicated in FIGS. 5, 6A and B.

[0209] In live brain tumor cell lines derived from human glioblastomas, Nox 4 was always expressed, along with variable expression of Nox 5 and gp91phox (FIG. 6B). Real time PCR revealed that the ratio of expression of Nox to G3PDH varied significantly in the various tumor cell lines compared with primary human astocytes (FIG. 6C). Although the cellular origin of glioblastomas has not been definitively established, this cancer type is thought by many workers to have arisen from the astrocytic lineage.

[0210] The expression of Nox forms in cancer and transformed cell lines did not correlate strictly with the expression in normal tissue, indicating that expression of Nox isoforms is sometimes altered in cancer cells. Thus, aberrant expression or regulation of Nox isoforms could account for the increased reactive oxygen generation seen in some cancer cells.

[0211] All patents, publications and abstracts cited above are incorporated herein by reference in their entirety. U.S. provisional patent applications serial Nos. 60/249,305, 60/251,364, 60/289,172, 60/289,537 are hereby incorporated by reference in their entirety. It should be understood that the foregoing relates only to preferred embodiments of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the present invention as defined in the following claims.

1 26 1 2232 DNA Homo sapiens CDS (87)..(1823) 1 ccgcacaact gtaaccgctg ccccggccgc cgcccgctcc ttctcgggcc ggcgggcaca 60 gagcgcagcg cggcggggcc ggcggc atg gct gtg tcc tgg agg agc tgg ctc 113 Met Ala Val Ser Trp Arg Ser Trp Leu 1 5 gcc aac gaa ggg gtt aaa cac ctc tgc ctg ttc atc tgg ctc tcc atg 161 Ala Asn Glu Gly Val Lys His Leu Cys Leu Phe Ile Trp Leu Ser Met 10 15 20 25 aat gtc ctg ctt ttc tgg aaa acc ttc ttg ctg tat aac caa ggg cca 209 Asn Val Leu Leu Phe Trp Lys Thr Phe Leu Leu Tyr Asn Gln Gly Pro 30 35 40 gag tat cac tac ctc cac cag atg ttg ggg cta gga ttg tgt cta agc 257 Glu Tyr His Tyr Leu His Gln Met Leu Gly Leu Gly Leu Cys Leu Ser 45 50 55 aga gcc tca gca tct gtt ctt aac ctc aac tgc agc ctt atc ctt tta 305 Arg Ala Ser Ala Ser Val Leu Asn Leu Asn Cys Ser Leu Ile Leu Leu 60 65 70 ccc atg tgc cga aca ctc ttg gct tac ctc cga gga tca cag aag gtt 353 Pro Met Cys Arg Thr Leu Leu Ala Tyr Leu Arg Gly Ser Gln Lys Val 75 80 85 cca agc agg aga acc agg aga ttg ttg gat aaa agc aga aca ttc cat 401 Pro Ser Arg Arg Thr Arg Arg Leu Leu Asp Lys Ser Arg Thr Phe His 90 95 100 105 att acc tgt ggt gtt act atc tgt att ttc tca ggc gtg cat gtg gct 449 Ile Thr Cys Gly Val Thr Ile Cys Ile Phe Ser Gly Val His Val Ala 110 115 120 gcc cat ctg gtg aat gcc ctc aac ttc tca gtg aat tac agt gaa gac 497 Ala His Leu Val Asn Ala Leu Asn Phe Ser Val Asn Tyr Ser Glu Asp 125 130 135 ttt gtt gaa ctg aat gca gca aga tac cga gat gag gat cct aga aaa 545 Phe Val Glu Leu Asn Ala Ala Arg Tyr Arg Asp Glu Asp Pro Arg Lys 140 145 150 ctt ctc ttc aca act gtt cct ggc ctg aca ggg gtc tgc atg gtg gtg 593 Leu Leu Phe Thr Thr Val Pro Gly Leu Thr Gly Val Cys Met Val Val 155 160 165 gtg cta ttc ctc atg atc aca gcc tct aca tat gca ata aga gtt tct 641 Val Leu Phe Leu Met Ile Thr Ala Ser Thr Tyr Ala Ile Arg Val Ser 170 175 180 185 aac tat gat atc ttc tgg tat act cat aac ctc ttc ttt gtc ttc tac 689 Asn Tyr Asp Ile Phe Trp Tyr Thr His Asn Leu Phe Phe Val Phe Tyr 190 195 200 atg ctg ctg acg ttg cat gtt tca gga ggg ctg ctg aag tat caa act 737 Met Leu Leu Thr Leu His Val Ser Gly Gly Leu Leu Lys Tyr Gln Thr 205 210 215 aat tta gat acc cac cct ccc ggc tgc atc agt ctt aac cga acc agc 785 Asn Leu Asp Thr His Pro Pro Gly Cys Ile Ser Leu Asn Arg Thr Ser 220 225 230 tct cag aat att tcc tta cca gag tat ttc tca gaa cat ttt cat gaa 833 Ser Gln Asn Ile Ser Leu Pro Glu Tyr Phe Ser Glu His Phe His Glu 235 240 245 cct ttc cct gaa gga ttt tca aaa ccg gca gag ttt acc cag cac aaa 881 Pro Phe Pro Glu Gly Phe Ser Lys Pro Ala Glu Phe Thr Gln His Lys 250 255 260 265 ttt gtg aag att tgt atg gaa gag ccc aga ttc caa gct aat ttt cca 929 Phe Val Lys Ile Cys Met Glu Glu Pro Arg Phe Gln Ala Asn Phe Pro 270 275 280 cag act tgg ctt tgg att tct gga cct ttg tgc ctg tac tgt gcc gaa 977 Gln Thr Trp Leu Trp Ile Ser Gly Pro Leu Cys Leu Tyr Cys Ala Glu 285 290 295 aga ctt tac agg tat atc cgg agc aat aag cca gtc acc atc att tcg 1025 Arg Leu Tyr Arg Tyr Ile Arg Ser Asn Lys Pro Val Thr Ile Ile Ser 300 305 310 gtc ata agt cat ccc tca gat gtc atg gaa atc cga atg gtc aaa gaa 1073 Val Ile Ser His Pro Ser Asp Val Met Glu Ile Arg Met Val Lys Glu 315 320 325 aat ttt aaa gca aga cct ggt cag tat att act cta cat tgt ccc agt 1121 Asn Phe Lys Ala Arg Pro Gly Gln Tyr Ile Thr Leu His Cys Pro Ser 330 335 340 345 gta tct gca tta gaa aat cat cca ttt acc ctc aca atg tgt cca act 1169 Val Ser Ala Leu Glu Asn His Pro Phe Thr Leu Thr Met Cys Pro Thr 350 355 360 gaa acc aaa gca aca ttt ggg gtt cat ctt aaa ata gta gga gac tgg 1217 Glu Thr Lys Ala Thr Phe Gly Val His Leu Lys Ile Val Gly Asp Trp 365 370 375 aca gaa cga ttt cga gat tta cta ctg cct cca tct agt caa gac tcc 1265 Thr Glu Arg Phe Arg Asp Leu Leu Leu Pro Pro Ser Ser Gln Asp Ser 380 385 390 gaa att ctg ccc ttc att caa tct aga aat tat ccc aag ctg tat att 1313 Glu Ile Leu Pro Phe Ile Gln Ser Arg Asn Tyr Pro Lys Leu Tyr Ile 395 400 405 gat ggt cct ttt gga agt cca ttt gag gaa tca ctg aac tat gag gtc 1361 Asp Gly Pro Phe Gly Ser Pro Phe Glu Glu Ser Leu Asn Tyr Glu Val 410 415 420 425 agc ctc tgc gtg gct gga ggc att gga gta act cca ttt gca tca ata 1409 Ser Leu Cys Val Ala Gly Gly Ile Gly Val Thr Pro Phe Ala Ser Ile 430 435 440 ctc aac acc ctg ttg gat gac tgg aaa cca tac aag ctt aga aga cta 1457 Leu Asn Thr Leu Leu Asp Asp Trp Lys Pro Tyr Lys Leu Arg Arg Leu 445 450 455 tac ttt att tgg gta tgc aga gat atc cag tcc ttc cgt tgg ttt gca 1505 Tyr Phe Ile Trp Val Cys Arg Asp Ile Gln Ser Phe Arg Trp Phe Ala 460 465 470 gat tta ctc tgt atg ttg cat aac aag ttt tgg caa gag aac aga cct 1553 Asp Leu Leu Cys Met Leu His Asn Lys Phe Trp Gln Glu Asn Arg Pro 475 480 485 gac tat gtc aac atc cag ctg tac ctc agt caa aca gat ggg ata cag 1601 Asp Tyr Val Asn Ile Gln Leu Tyr Leu Ser Gln Thr Asp Gly Ile Gln 490 495 500 505 aag ata att gga gaa aaa tat cat gca ctg aat tca aga ctg ttt ata 1649 Lys Ile Ile Gly Glu Lys Tyr His Ala Leu Asn Ser Arg Leu Phe Ile 510 515 520 gga cgt cct cgg tgg aaa ctt ttg ttt gat gaa ata gca aaa tat aac 1697 Gly Arg Pro Arg Trp Lys Leu Leu Phe Asp Glu Ile Ala Lys Tyr Asn 525 530 535 aga gga aaa aca gtt ggt gtt ttc tgt tgt gga ccc aat tca cta tcc 1745 Arg Gly Lys Thr Val Gly Val Phe Cys Cys Gly Pro Asn Ser Leu Ser 540 545 550 aag act ctt cat aaa ctg agt aac cag aac aac tca tat ggg aca aga 1793 Lys Thr Leu His Lys Leu Ser Asn Gln Asn Asn Ser Tyr Gly Thr Arg 555 560 565 ttt gaa tac aat aaa gag tct ttc agc tga aaacttttgc catgaagcag 1843 Phe Glu Tyr Asn Lys Glu Ser Phe Ser 570 575 gactctaaag aaggaatgag tgcaatttct aagactttga aactcagcgg aatcaatcag 1903 ctgtgttatg ccaaagaata gtaaggtttt cttatttatg attatttgaa aatggaaatg 1963 tgagaatgtg gcaacatgac cgtcacatta catgtttaat ctggaaacca aagagaccct 2023 gaagaatatt tgatgtgatg attcattttc agttctcaaa ttaaaagaaa actgttagat 2083 gcacactgtt gattttcatg gtggattcaa gaactcccta gtgaggagct gaacttgctc 2143 aatctaaggc tgattgtcgt gttcctcttt aaattgtttt tggttgaaca aatgcaagat 2203 tgaacaaaat taaaaattca ttgaagctg 2232 2 578 PRT Homo sapiens 2 Met Ala Val Ser Trp Arg Ser Trp Leu Ala Asn Glu Gly Val Lys His 1 5 10 15 Leu Cys Leu Phe Ile Trp Leu Ser Met Asn Val Leu Leu Phe Trp Lys 20 25 30 Thr Phe Leu Leu Tyr Asn Gln Gly Pro Glu Tyr His Tyr Leu His Gln 35 40 45 Met Leu Gly Leu Gly Leu Cys Leu Ser Arg Ala Ser Ala Ser Val Leu 50 55 60 Asn Leu Asn Cys Ser Leu Ile Leu Leu Pro Met Cys Arg Thr Leu Leu 65 70 75 80 Ala Tyr Leu Arg Gly Ser Gln Lys Val Pro Ser Arg Arg Thr Arg Arg 85 90 95 Leu Leu Asp Lys Ser Arg Thr Phe His Ile Thr Cys Gly Val Thr Ile 100 105 110 Cys Ile Phe Ser Gly Val His Val Ala Ala His Leu Val Asn Ala Leu 115 120 125 Asn Phe Ser Val Asn Tyr Ser Glu Asp Phe Val Glu Leu Asn Ala Ala 130 135 140 Arg Tyr Arg Asp Glu Asp Pro Arg Lys Leu Leu Phe Thr Thr Val Pro 145 150 155 160 Gly Leu Thr Gly Val Cys Met Val Val Val Leu Phe Leu Met Ile Thr 165 170 175 Ala Ser Thr Tyr Ala Ile Arg Val Ser Asn Tyr Asp Ile Phe Trp Tyr 180 185 190 Thr His Asn Leu Phe Phe Val Phe Tyr Met Leu Leu Thr Leu His Val 195 200 205 Ser Gly Gly Leu Leu Lys Tyr Gln Thr Asn Leu Asp Thr His Pro Pro 210 215 220 Gly Cys Ile Ser Leu Asn Arg Thr Ser Ser Gln Asn Ile Ser Leu Pro 225 230 235 240 Glu Tyr Phe Ser Glu His Phe His Glu Pro Phe Pro Glu Gly Phe Ser 245 250 255 Lys Pro Ala Glu Phe Thr Gln His Lys Phe Val Lys Ile Cys Met Glu 260 265 270 Glu Pro Arg Phe Gln Ala Asn Phe Pro Gln Thr Trp Leu Trp Ile Ser 275 280 285 Gly Pro Leu Cys Leu Tyr Cys Ala Glu Arg Leu Tyr Arg Tyr Ile Arg 290 295 300 Ser Asn Lys Pro Val Thr Ile Ile Ser Val Ile Ser His Pro Ser Asp 305 310 315 320 Val Met Glu Ile Arg Met Val Lys Glu Asn Phe Lys Ala Arg Pro Gly 325 330 335 Gln Tyr Ile Thr Leu His Cys Pro Ser Val Ser Ala Leu Glu Asn His 340 345 350 Pro Phe Thr Leu Thr Met Cys Pro Thr Glu Thr Lys Ala Thr Phe Gly 355 360 365 Val His Leu Lys Ile Val Gly Asp Trp Thr Glu Arg Phe Arg Asp Leu 370 375 380 Leu Leu Pro Pro Ser Ser Gln Asp Ser Glu Ile Leu Pro Phe Ile Gln 385 390 395 400 Ser Arg Asn Tyr Pro Lys Leu Tyr Ile Asp Gly Pro Phe Gly Ser Pro 405 410 415 Phe Glu Glu Ser Leu Asn Tyr Glu Val Ser Leu Cys Val Ala Gly Gly 420 425 430 Ile Gly Val Thr Pro Phe Ala Ser Ile Leu Asn Thr Leu Leu Asp Asp 435 440 445 Trp Lys Pro Tyr Lys Leu Arg Arg Leu Tyr Phe Ile Trp Val Cys Arg 450 455 460 Asp Ile Gln Ser Phe Arg Trp Phe Ala Asp Leu Leu Cys Met Leu His 465 470 475 480 Asn Lys Phe Trp Gln Glu Asn Arg Pro Asp Tyr Val Asn Ile Gln Leu 485 490 495 Tyr Leu Ser Gln Thr Asp Gly Ile Gln Lys Ile Ile Gly Glu Lys Tyr 500 505 510 His Ala Leu Asn Ser Arg Leu Phe Ile Gly Arg Pro Arg Trp Lys Leu 515 520 525 Leu Phe Asp Glu Ile Ala Lys Tyr Asn Arg Gly Lys Thr Val Gly Val 530 535 540 Phe Cys Cys Gly Pro Asn Ser Leu Ser Lys Thr Leu His Lys Leu Ser 545 550 555 560 Asn Gln Asn Asn Ser Tyr Gly Thr Arg Phe Glu Tyr Asn Lys Glu Ser 565 570 575 Phe Ser 3 2223 DNA Homo sapiens CDS (73)..(1770) 3 gccgacgcgg acggcaacgg ggccatcacc ttcgaggagc tccgggacga gctgcagcgc 60 ttccccggag tc atg gag aac ctg acc atc agc act gcc cac tgg ctg acg 111 Met Glu Asn Leu Thr Ile Ser Thr Ala His Trp Leu Thr 1 5 10 gcc ccc gcc ccc cgc cca cgc ccg cgc cgg ccg cgc cag ctg acc cgc 159 Ala Pro Ala Pro Arg Pro Arg Pro Arg Arg Pro Arg Gln Leu Thr Arg 15 20 25 gcc tac tgg cac aac cac cgc agc cag ctg ttc tgc ctg gcc acc tat 207 Ala Tyr Trp His Asn His Arg Ser Gln Leu Phe Cys Leu Ala Thr Tyr 30 35 40 45 gca ggc ctc cac gtg ctg ctc ttc ggg ctg gcg gcc agc gcg cac cgg 255 Ala Gly Leu His Val Leu Leu Phe Gly Leu Ala Ala Ser Ala His Arg 50 55 60 gac ctc ggc gcc agc gtc atg gtg gcc aag ggc tgt ggc cag tgc ctc 303 Asp Leu Gly Ala Ser Val Met Val Ala Lys Gly Cys Gly Gln Cys Leu 65 70 75 aac ttc gac tgc agc ttc atc gcg gtg ctg atg ctc aga cgc tgc ctc 351 Asn Phe Asp Cys Ser Phe Ile Ala Val Leu Met Leu Arg Arg Cys Leu 80 85 90 acc tgg ctg cgg gcc acg tgg ctg gct caa gtc cta cca ctg gac cag 399 Thr Trp Leu Arg Ala Thr Trp Leu Ala Gln Val Leu Pro Leu Asp Gln 95 100 105 aac atc cag ttc cac cag ctt atg ggc tac gtg gta gtg ggg ctg tcc 447 Asn Ile Gln Phe His Gln Leu Met Gly Tyr Val Val Val Gly Leu Ser 110 115 120 125 ctc gtg cac act gtg gct cac act gtg aac ttt gta ctc cag gct cag 495 Leu Val His Thr Val Ala His Thr Val Asn Phe Val Leu Gln Ala Gln 130 135 140 gcg gag gcc agc cct ttc cag ttc tgg gag ctg ctg ctc acc acg agg 543 Ala Glu Ala Ser Pro Phe Gln Phe Trp Glu Leu Leu Leu Thr Thr Arg 145 150 155 cct ggc att ggc tgg gta cac ggt tcg gcc tcc ccg aca ggt gtc gct 591 Pro Gly Ile Gly Trp Val His Gly Ser Ala Ser Pro Thr Gly Val Ala 160 165 170 ctg ctg ctg ctg ctc ctc ctc atg ttc atc tgc tcc agt tcc tgc atc 639 Leu Leu Leu Leu Leu Leu Leu Met Phe Ile Cys Ser Ser Ser Cys Ile 175 180 185 cgc agg agt ggc cac ttt gag gtg ttc tat tgg act cac ctg tcc tac 687 Arg Arg Ser Gly His Phe Glu Val Phe Tyr Trp Thr His Leu Ser Tyr 190 195 200 205 ctc ctc gtg tgg ctt ctg ctc atc ttt cat ggg ccc aac ttc tgg aag 735 Leu Leu Val Trp Leu Leu Leu Ile Phe His Gly Pro Asn Phe Trp Lys 210 215 220 tgg ctg ctg gtg cct gga atc ttg ttt ttc ctg gag aag gcc atc gga 783 Trp Leu Leu Val Pro Gly Ile Leu Phe Phe Leu Glu Lys Ala Ile Gly 225 230 235 ctg gca gtg tcc cgc atg gca gcc gtg tgc atc atg gaa gtc aac ctc 831 Leu Ala Val Ser Arg Met Ala Ala Val Cys Ile Met Glu Val Asn Leu 240 245 250 ctc ccc tcc aag gtc act cat ctc ctc atc aag cgg ccc cct ttt ttt 879 Leu Pro Ser Lys Val Thr His Leu Leu Ile Lys Arg Pro Pro Phe Phe 255 260 265 cac tat aga cct ggt gac tac ttg tat ctg aac atc ccc acc att gct 927 His Tyr Arg Pro Gly Asp Tyr Leu Tyr Leu Asn Ile Pro Thr Ile Ala 270 275 280 285 cgc tat gag tgg cac ccc ttc acc atc agc agt gct cct gag cag aaa 975 Arg Tyr Glu Trp His Pro Phe Thr Ile Ser Ser Ala Pro Glu Gln Lys 290 295 300 gac act atc tgg ctg cac att cgg tcc caa ggc cag tgg aca aac agg 1023 Asp Thr Ile Trp Leu His Ile Arg Ser Gln Gly Gln Trp Thr Asn Arg 305 310 315 ctg tat gag tcc ttc aag gca tca gac cca ctg ggc cgt ggt tct aag 1071 Leu Tyr Glu Ser Phe Lys Ala Ser Asp Pro Leu Gly Arg Gly Ser Lys 320 325 330 agg ctg tcg agg agt gtg aca atg aga aag agt caa agg tcg tcc aag 1119 Arg Leu Ser Arg Ser Val Thr Met Arg Lys Ser Gln Arg Ser Ser Lys 335 340 345 ggc tct gag ata ctt ttg gag aaa cac aaa ttc tgt aac atc aag tgc 1167 Gly Ser Glu Ile Leu Leu Glu Lys His Lys Phe Cys Asn Ile Lys Cys 350 355 360 365 tac atc gat ggg cct tat ggg acc ccc acc cgc agg atc ttt gcc tct 1215 Tyr Ile Asp Gly Pro Tyr Gly Thr Pro Thr Arg Arg Ile Phe Ala Ser 370 375 380 gag cat gcc gtg ctc atc ggg gca ggc atc ggc atc acc ccc ttt gct 1263 Glu His Ala Val Leu Ile Gly Ala Gly Ile Gly Ile Thr Pro Phe Ala 385 390 395 tcc att ctg cag agt atc atg tac agg cac cag aaa aga aag cat act 1311 Ser Ile Leu Gln Ser Ile Met Tyr Arg His Gln Lys Arg Lys His Thr 400 405 410 tgc ccc agc tgc cag cac tcc tgg atc gaa ggt gtc caa gac aac atg 1359 Cys Pro Ser Cys Gln His Ser Trp Ile Glu Gly Val Gln Asp Asn Met 415 420 425 aag ctc cat aag gtg gac ttt atc tgg atc aac aga gac cag cgg tct 1407 Lys Leu His Lys Val Asp Phe Ile Trp Ile Asn Arg Asp Gln Arg Ser 430 435 440 445 ttc gag tgg ttt gtg agc ctg ctg act aaa ctg gag atg gac cag gcc 1455 Phe Glu Trp Phe Val Ser Leu Leu Thr Lys Leu Glu Met Asp Gln Ala 450 455 460 gag gag gct caa tac ggc cgc ttc ctg gag ctg cat atg tac atg aca 1503 Glu Glu Ala Gln Tyr Gly Arg Phe Leu Glu Leu His Met Tyr Met Thr 465 470 475 tct gca ctg ggc aag aat gac atg aag gcc att ggc ctg cag atg gcc 1551 Ser Ala Leu Gly Lys Asn Asp Met Lys Ala Ile Gly Leu Gln Met Ala 480 485 490 ctt gac ctc ctg gcc aac aag gag aag aaa gac tcc atc acg ggg ctg 1599 Leu Asp Leu Leu Ala Asn Lys Glu Lys Lys Asp Ser Ile Thr Gly Leu 495 500 505 cag acg cgc acc cag cct ggg cgg cct gac tgg agc aag gtg ttc cag 1647 Gln Thr Arg Thr Gln Pro Gly Arg Pro Asp Trp Ser Lys Val Phe Gln 510 515 520 525 aaa gtg gct gct gag aag aag ggc aag gtg cag gtc ttc ttc tgt ggc 1695 Lys Val Ala Ala Glu Lys Lys Gly Lys Val Gln Val Phe Phe Cys Gly 530 535 540 tcc cca gct ctg gcc aag gtg ctg aag ggc cat tgt gag aag ttc ggc 1743 Ser Pro Ala Leu Ala Lys Val Leu Lys Gly His Cys Glu Lys Phe Gly 545 550 555 ttc aga ttt ttc caa gag aat ttc tag cctcacctct ccaagctctg 1790 Phe Arg Phe Phe Gln Glu Asn Phe 560 565 ccccaagtcc acaccatggg tctgcttcat cgcattagta taaatgcccc cacagggacc 1850 agcctcagat gacccaccca ataagacaaa gcctagggac cccctaatcc tgctcaacag 1910 agagaacagg agaccccaag gggcagatga acttcctcta gaacccaggg gaaggggcag 1970 tgccttgttc agtctgctgt agattctggg gtttctgtga aagtgaggga accagaggct 2030 ggtcacggga gcttgggggt ggggttcgag ggggcagagg gcaaccactc ctccaaacat 2090 tttccgacgg agccttcccc cacatccatg gtcccaaacc tgcccaatca tcacagtcat 2150 ttggaagctt atttctccgg catcttataa aattgttcaa acctacagta aaaaaaaaaa 2210 aaaaaaaaaa aaa 2223 4 565 PRT Homo sapiens 4 Met Glu Asn Leu Thr Ile Ser Thr Ala His Trp Leu Thr Ala Pro Ala 1 5 10 15 Pro Arg Pro Arg Pro Arg Arg Pro Arg Gln Leu Thr Arg Ala Tyr Trp 20 25 30 His Asn His Arg Ser Gln Leu Phe Cys Leu Ala Thr Tyr Ala Gly Leu 35 40 45 His Val Leu Leu Phe Gly Leu Ala Ala Ser Ala His Arg Asp Leu Gly 50 55 60 Ala Ser Val Met Val Ala Lys Gly Cys Gly Gln Cys Leu Asn Phe Asp 65 70 75 80 Cys Ser Phe Ile Ala Val Leu Met Leu Arg Arg Cys Leu Thr Trp Leu 85 90 95 Arg Ala Thr Trp Leu Ala Gln Val Leu Pro Leu Asp Gln Asn Ile Gln 100 105 110 Phe His Gln Leu Met Gly Tyr Val Val Val Gly Leu Ser Leu Val His 115 120 125 Thr Val Ala His Thr Val Asn Phe Val Leu Gln Ala Gln Ala Glu Ala 130 135 140 Ser Pro Phe Gln Phe Trp Glu Leu Leu Leu Thr Thr Arg Pro Gly Ile 145 150 155 160 Gly Trp Val His Gly Ser Ala Ser Pro Thr Gly Val Ala Leu Leu Leu 165 170 175 Leu Leu Leu Leu Met Phe Ile Cys Ser Ser Ser Cys Ile Arg Arg Ser 180 185 190 Gly His Phe Glu Val Phe Tyr Trp Thr His Leu Ser Tyr Leu Leu Val 195 200 205 Trp Leu Leu Leu Ile Phe His Gly Pro Asn Phe Trp Lys Trp Leu Leu 210 215 220 Val Pro Gly Ile Leu Phe Phe Leu Glu Lys Ala Ile Gly Leu Ala Val 225 230 235 240 Ser Arg Met Ala Ala Val Cys Ile Met Glu Val Asn Leu Leu Pro Ser 245 250 255 Lys Val Thr His Leu Leu Ile Lys Arg Pro Pro Phe Phe His Tyr Arg 260 265 270 Pro Gly Asp Tyr Leu Tyr Leu Asn Ile Pro Thr Ile Ala Arg Tyr Glu 275 280 285 Trp His Pro Phe Thr Ile Ser Ser Ala Pro Glu Gln Lys Asp Thr Ile 290 295 300 Trp Leu His Ile Arg Ser Gln Gly Gln Trp Thr Asn Arg Leu Tyr Glu 305 310 315 320 Ser Phe Lys Ala Ser Asp Pro Leu Gly Arg Gly Ser Lys Arg Leu Ser 325 330 335 Arg Ser Val Thr Met Arg Lys Ser Gln Arg Ser Ser Lys Gly Ser Glu 340 345 350 Ile Leu Leu Glu Lys His Lys Phe Cys Asn Ile Lys Cys Tyr Ile Asp 355 360 365 Gly Pro Tyr Gly Thr Pro Thr Arg Arg Ile Phe Ala Ser Glu His Ala 370 375 380 Val Leu Ile Gly Ala Gly Ile Gly Ile Thr Pro Phe Ala Ser Ile Leu 385 390 395 400 Gln Ser Ile Met Tyr Arg His Gln Lys Arg Lys His Thr Cys Pro Ser 405 410 415 Cys Gln His Ser Trp Ile Glu Gly Val Gln Asp Asn Met Lys Leu His 420 425 430 Lys Val Asp Phe Ile Trp Ile Asn Arg Asp Gln Arg Ser Phe Glu Trp 435 440 445 Phe Val Ser Leu Leu Thr Lys Leu Glu Met Asp Gln Ala Glu Glu Ala 450 455 460 Gln Tyr Gly Arg Phe Leu Glu Leu His Met Tyr Met Thr Ser Ala Leu 465 470 475 480 Gly Lys Asn Asp Met Lys Ala Ile Gly Leu Gln Met Ala Leu Asp Leu 485 490 495 Leu Ala Asn Lys Glu Lys Lys Asp Ser Ile Thr Gly Leu Gln Thr Arg 500 505 510 Thr Gln Pro Gly Arg Pro Asp Trp Ser Lys Val Phe Gln Lys Val Ala 515 520 525 Ala Glu Lys Lys Gly Lys Val Gln Val Phe Phe Cys Gly Ser Pro Ala 530 535 540 Leu Ala Lys Val Leu Lys Gly His Cys Glu Lys Phe Gly Phe Arg Phe 545 550 555 560 Phe Gln Glu Asn Phe 565 5 3999 DNA Homo sapiens 5 tgccaatttc acctggcatt atgatactaa attgcaggtg tgttcagcag ctaatgaaaa 60 tgttgttttt atgggttttg tgatgttgaa gagatttttc agctttttac aataaatata 120 aaatgtgctg tgcattgcgc tatactcctt ataatgggaa catttcatgg catataaatt 180 atacctcaat aaatctgtta attgtgctgt gctttctttt ctgttttaaa taaatatttg 240 cttttgtgcc taaagtaaag taaaataaaa atatgtagca gtatatctac aaaacctata 300 caaaacattc tacttaaggg tgaaatggaa gaatcccctt tgagattggg aattaggcaa 360 ggggatcact acttctacct ttgtgcagca ttgtactaga agttctagtg agtgcagtaa 420 gcgaagaaaa agaaataaaa gttataagaa ttagaaaaaa gaaggctggg cacggtggct 480 cacgcctgta atcccagcat tttgggaggc cgaggcaggc gtatcacctg aggtcaggtg 540 tttcagacct acctggccaa catggtaaaa cccagtctct actaaaaata gaaaaattag 600 ccgagcatgg tggtgggcgc ctgtagtccc agctactcgg gaggctgagg caggagaatc 660 gcttgaaccc gggaggcaga cgttgcagta agccattgca ctccagcctg ggcaacaaga 720 gggaaactcc gtctcaaaaa aaaaaaaaag aattagaaaa aagaaataaa actgtgttta 780 gagcgaaaaa tgtgactgtg tttatagaca atgtaagaca aaatacagaa atattattac 840 aattaataag tgagtttagc aatgttgctg ggcataaaaa tcaatatgta aaaacaaatt 900 gtatctttaa cagccatcat taaaaattaa aattaaaata tataatttac aacctcatga 960 aaacatataa agttcttaga agtggatata acaggctggg cacggtggct catgcctgta 1020 atcccagcac tttgggaggc cgaggcaggc ggatcacgag gtgaggaggt cgagaccatc 1080 ctggctagca cagtgaaacc ccgtctctac taaaaaatag aaaaaattag ccaggcgtgg 1140 tggcgggcgc ctgtggtccc agctactcgg gaggctgagg caggagaatg gcgtgaaccc 1200 gggaggtgga gcttgcagtg agccgagatt gcaccactgc actccagcct gggcgacaga 1260 gcgagactcc gtctcaaaga aaaaaaaaaa agaagaagaa gtgaatataa caaaggagac 1320 gcaagaattt gattataaaa tttacatgaa aatgaaaaag gccaagaata aaaacaaaac 1380 acagaacccc ccctcccccc aaaacaaaga aacaaacaaa caaaaagtca agaatagcca 1440 aagcactctt gaagaacaag gtggggaaac ttgtcttatc agatgtcaag acttagtaat 1500 taaggcagtg gcatggaaga actgaatgga gagctccgga agaaactcgt gtatatagac 1560 atttcatata taatagaact gacattttag atcagcgaag aaagtatatt tttgaaagta 1620 cttttcaaaa atggctgagc gcagtggctc atgcctgtaa tcccagcact ttgagaggcc 1680 cacgtgggtg gatcacatga ggccaggagg tcaagaccag actggccagc acagcaaaac 1740 cctgtctcta ctaaaaatac aaaaattagc caggcatgat ggcgcttgcc tgtaatccca 1800 gctactcggg tggctaaggc atgagaatca cttgaaccca ggaggtgaag gttgcagtga 1860 agggagatca caccactgta ctccaacctg ggtgacagag tgagaccctg tctcaaaaaa 1920 ataaaaataa aaataaaaag tacttttcaa aaatgatgct ggggcaattc gttttccata 1980 tttaaaagta gtaaattgga taccatacat gaaaatcagc tccaggtgga ttcaaaacat 2040 aaatgtaaaa tgcaaaaata taaaatttct agaagaaaat ataaaagagt atcttgatat 2100 ctgggtagtg atggatttct aaaacaagac ataaaatgca taaatcataa aagaaatgac 2160 tggtaatcag agtgcattaa aattaagaac ttccatttat cagaaaacac tattaagaga 2220 ctgaaaagac aagccataaa cataagcaat aaaagattag tataagatta taaacagaac 2280 cctaagaatc taaaagcaaa agaaaaacca atagaaagat agaccaaaaa gtagaatagg 2340 ctcagaatag gctcttttaa aaagagaaaa ctcaaatggc cagcagttga attaaaagat 2400 gctcaaactc attagtaatc agggaaatgc aaattaaaat cataatacga tagttttcca 2460 cacttacttg aattataaaa acaaaaaagt ctggaaaata ccaagggttg gtaagcatgt 2520 agaggaagta gaactctcat tcataactct ctgtagtata catttaggtg gtcacttcgg 2580 aacgggtttg gaattacaca gcaaagtaga atatgtgcaa atctcaggac cctggaattt 2640 tactcctggg tatatacctt agagaaactg tagcatatgt gtgacatttg atcaacattg 2700 ttccatcatc atatccatca gtagtaggat gaatgaatac attaatgtat attcattatg 2760 caatggcata ttagatagca gtgtaagtga accgcaatta catgtacatg tatgaatctc 2820 aaaaacccaa tgttgaaaga agcaaaccac agaagcatac atacacactg ccaggtttca 2880 tttacaaaaa gttcaaaaac aggaaaaact aaacaatata ttgcttaggg atgcaattat 2940 agttagtaaa aatataaaga aaaataacag aatgattacc ccaaatttca ggatagtgat 3000 tacatccggt ggggtagagg aggggaagaa gatagatgtg atcagggagg gaaatacaaa 3060 gagctttaag atactggaga aaaatagtct attttcttta atctgagtag tgaacacata 3120 gatacttatt ccttaaaatt attctttaag ttacatatgt atgttttata tactcttctg 3180 tgtatatttc accattttag aaaagggaaa aaaaatcagt gcccagagct gaacacacaa 3240 ctctagtaaa tctatcatac tagaagacaa tcatctccat tcttttgagt gctctgcctc 3300 tgtttatttt gaaccaaagt gcacttttat acttgttaaa ttttctcttg ctctatttgg 3360 cccttctttt cacttgtcct tccagccagt caagttctcc ccaaagccat catcatatat 3420 gtcaaccaca gatcatcctc caggggaact ggtatgctaa agtttctgag ctagccaggc 3480 tgaaatccaa atggcagccg gcagatgtgg caacagtttg aaaagtgcac tttgaaacag 3540 cttccttacc acacacgctt ccctccctac ttctcctgaa gtaatctgtt tacagaccca 3600 gactaataat cttttttatg agaaacttta gcaaatcttt tatctaggaa ggcaatgctt 3660 cacattaggt catgttgata agatgatgag agagaatatt ttcatccaag aatgttgcta 3720 tttcctgaag cagtaaaatc cccacaggta aaacccttgt ggttctcata gatagggctg 3780 gtctatctaa gctgatagca cagttctgtc cagagaagga aggcagaata aacttattca 3840 ttcccaggaa ctcttggggt aggtgtgtgt ttttcacatc ttaaaggctc acagaccctg 3900 cgctggacaa atgttccatt cctgaaggac ctctccagaa tccggattgc tgaatcttcc 3960 ctgttgccta gaagggctcc aaaccacctc ttgacaatg 3999 6 2044 DNA Homo sapiens 6 caaagacaaa ataatttact agggaagccc ttactaacga cccaacatcc agacacaggt 60 gagggagaag aaatttcctg acagccgaag agcaacaagt atcatgatgg ggtgctggat 120 tttgaatgag ggtctctcca ccatattagt actctcatgg ctgggaataa atttttatct 180 gtttattgac acgttctact ggtatgaaga ggaggagtct ttccattaca cacgagttat 240 tttgggttca acactggctt gggcacgagc atccgcactg tgcctgaatt ttaactgcat 300 gctaattcta atacctgtca gtcgaaacct tatttcattc ataagaggaa caagtatttg 360 ctgcagagga ccgtggagga ggcaattaga caaaaacctc agatttcaca aactggtcgc 420 ctatgggata gctgttaatg caaccatcca catcgtggcg catttcttca acctggaacg 480 ctaccactgg agccagtccg aggaggccca gggacttctg gccgcacttt ccaagctggg 540 caacacccct aacgagagct acctcaaccc tgtccggacc ttccccacaa acacaaccac 600 tgaattgcta aggacaatag caggcgtcac cggtctggtg atctctctgg ctttagtctt 660 gatcatgacc tcgtcaactg agttcatcag acaggcctcc tatgagttgt tctggtacac 720 acaccatgtt ttcatcgtct tctttctcag cctggccatc catgggacgg gtcggattgt 780 tcgaggccaa acccaagaca gtctctctct gcacaacatc accttctgta gagaccgcta 840 tgcagaatgg cagacagtgg cccaatgccc cgtgcctcaa ttttctggca aggaaccctc 900 ggcttggaaa tggattttag gccctgtggt cttgtatgca tgtgaaagaa taattaggtt 960 ctggcgattt caacaagaag ttgtcattac caaggtggta agccacccct ctggagtcct 1020 ggaacttcac atgaaaaagc gtggctttaa aatggcgcca gggcagtaca tcttggtgca 1080 gtgcccagcc atatcttcgc tggagtggca ccccttcacc cttacctctg ccccccagga 1140 agactttttc agcgtgcaca tccgggcagc aggagactgg acagcagcgc tactggaggc 1200 ctttggggca gagggacagg ccctccagga gccctggagc ctgccaaggc tggcagtgga 1260 cgggcccttt ggaactgccc tgacagatgt atttcactac ccagtgtgtg tgtgcgttgc 1320 cgcggggatc ggagtcactc ccttcgctgc tcttctgaaa tctatatggt acaaatgcag 1380 tgaggcacag accccactga agctgagcaa ggtgtatttc tactggattt gccgggatgc 1440 aagagctttt gagtggtttg ctgatctctt actctccctg gaaacacgga tgagtgagca 1500 ggggaaaact cactttctga gttatcatat atttcttacc ggctgggatg aaaatcaggc 1560 tcttcacata gctttacact gggacgaaaa tactgacgtg attacaggct taaagcagaa 1620 gaccttctat gggaggccca actggaacaa tgagttcaag cagattgcct acaatcaccc 1680 cagcagcagt attggcgtgt tcttctgtgg acctaaagct ctctcgagga cacttcaaaa 1740 gatgtgccac ttgtattcat cagctgaccc cagaggtgtt catttctatt acaacaagga 1800 gagcttctag actttggagg tcaagtccag gcattgtgtt ttcaatcaag ttattgattc 1860 caaagaactc caccaggaat tcctgtgacg gcctgttgat atgagctccc agttgggaac 1920 tggtgaataa taattaacta ttgtgaacag tacactatac catacttcct tagcttataa 1980 ataacatgtc atatacaaca gaacaaaaac atttactgaa attaaaatat attatgtttc 2040 tcca 2044 7 25 DNA Artificial Sequence Synthetic Primer 7 caacgaaggg gttaaacacc tctgc 25 8 23 DNA Artificial Sequence Synthetic Primer 8 cacagctgat tgattccgct gag 23 9 24 DNA Artificial Sequence Synthetic Primer 9 taagccaaga gtgttcggca catg 24 10 25 DNA Artificial Sequence Synthetic Primer 10 tactctggcc cttggttata cagca 25 11 22 DNA Artificial Sequence Synthetic Primer 11 tccatttacc ctcacaatgt gt 22 12 23 DNA Artificial Sequence Synthetic Primer 12 ctcagcggaa tcaatcagct gtg 23 13 569 PRT Homo sapiens 13 Gly Asn Trp Ala Val Asn Glu Gly Leu Ser Ile Phe Ala Ile Leu Val 1 5 10 15 Trp Leu Gly Leu Asn Val Phe Leu Phe Val Trp Tyr Tyr Arg Val Tyr 20 25 30 Asp Ile Pro Pro Lys Phe Phe Tyr Thr Arg Lys Leu Leu Gly Ser Ala 35 40 45 Leu Ala Leu Ala Arg Ala Pro Ala Ala Cys Leu Asn Phe Asn Cys Met 50 55 60 Leu Ile Leu Leu Pro Val Cys Arg Asn Leu Leu Ser Phe Leu Arg Gly 65 70 75 80 Ser Ser Ala Cys Cys Ser Thr Arg Val Arg Arg Gln Leu Asp Arg Asn 85 90 95 Leu Thr Phe His Lys Met Val Ala Trp Met Ile Ala Leu His Ser Ala 100 105 110 Ile His Thr Ile Ala His Leu Phe Asn Val Glu Trp Cys Val Asn Ala 115 120 125 Arg Val Asn Asn Ser Asp Pro Tyr Ser Val Ala Leu Ser Glu Leu Gly 130 135 140 Asp Arg Gln Asn Glu Ser Tyr Leu Asn Phe Ala Arg Lys Arg Ile Lys 145 150 155 160 Asn Pro Glu Gly Gly Leu Tyr Leu Ala Val Thr Leu Leu Ala Gly Ile 165 170 175 Thr Gly Val Val Ile Thr Leu Cys Leu Ile Leu Ile Ile Thr Ser Ser 180 185 190 Thr Lys Thr Ile Arg Arg Ser Tyr Phe Glu Val Phe Trp Tyr Thr His 195 200 205 His Leu Phe Val Ile Phe Phe Ile Gly Leu Ala Ile His Gly Ala Glu 210 215 220 Arg Ile Val Arg Gly Gln Thr Ala Glu Ser Leu Ala Val His Asn Ile 225 230 235 240 Thr Val Cys Glu Gln Lys Ile Ser Glu Trp Gly Lys Ile Lys Glu Cys 245 250 255 Pro Ile Pro Gln Phe Ala Gly Asn Pro Pro Met Thr Trp Lys Trp Ile 260 265 270 Val Gly Pro Met Phe Leu Tyr Leu Cys Glu Arg Leu Val Arg Phe Trp 275 280 285 Arg Ser Gln Gln Lys Val Val Ile Thr Lys Val Val Thr His Pro Phe 290 295 300 Lys Thr Ile Glu Leu Gln Met Lys Lys Lys Gly Phe Lys Met Glu Val 305 310 315 320 Gly Gln Tyr Ile Phe Val Lys Cys Pro Lys Val Ser Lys Leu Glu Trp 325 330 335 His Pro Phe Thr Leu Thr Ser Ala Pro Glu Glu Asp Phe Phe Ser Ile 340 345 350 His Ile Arg Ile Val Gly Asp Trp Thr Glu Gly Leu Phe Asn Ala Cys 355 360 365 Gly Cys Asp Lys Gln Glu Phe Gln Asp Ala Trp Lys Leu Pro Lys Ile 370 375 380 Ala Val Asp Gly Pro Phe Gly Thr Ala Ser Glu Asp Val Phe Ser Tyr 385 390 395 400 Glu Val Val Met Leu Val Gly Ala Gly Ile Gly Val Thr Pro Phe Ala 405 410 415 Ser Ile Leu Lys Ser Val Trp Tyr Lys Tyr Cys Asn Asn Ala Thr Asn 420 425 430 Leu Lys Leu Lys Lys Ile Tyr Phe Tyr Trp Leu Cys Arg Asp Thr His 435 440 445 Ala Phe Glu Trp Phe Ala Asp Leu Leu Gln Leu Leu Glu Ser Gln Met 450 455 460 Gln Glu Arg Asn Asn Ala Gly Phe Leu Ser Tyr Asn Ile Tyr Leu Thr 465 470 475 480 Gly Trp Asp Glu Ser Gln Ala Asn His Phe Ala Val His His Asp Glu 485 490 495 Glu Lys Asp Val Ile Thr Gly Leu Lys Gln Lys Thr Leu Tyr Gly Arg 500 505 510 Pro Asn Trp Asp Asn Glu Phe Lys Thr Ile Ala Ser Gln His Pro Asn 515 520 525 Thr Arg Ile Gly Val Phe Leu Cys Gly Pro Glu Ala Leu Ala Glu Thr 530 535 540 Leu Ser Lys Gln Ser Ile Ser Asn Ser Glu Ser Gly Pro Arg Gly Val 545 550 555 560 His Phe Ile Phe Asn Lys Glu Asn Phe 565 14 564 PRT Homo sapiens 14 Met Gly Asn Trp Val Val Asn His Trp Phe Ser Val Leu Phe Leu Val 1 5 10 15 Val Trp Leu Gly Leu Asn Val Phe Leu Phe Val Asp Ala Phe Leu Lys 20 25 30 Tyr Glu Lys Ala Asp Lys Tyr Tyr Tyr Thr Arg Lys Ile Leu Gly Ser 35 40 45 Thr Leu Ala Cys Ala Arg Ala Ser Ala Leu Cys Leu Asn Phe Asn Ser 50 55 60 Thr Leu Ile Leu Leu Pro Val Cys Arg Asn Leu Leu Ser Phe Leu Arg 65 70 75 80 Gly Thr Cys Ser Phe Cys Ser Arg Thr Leu Arg Lys Gln Leu Asp His 85 90 95 Asn Leu Thr Phe His Lys Leu Val Ala Tyr Met Ile Cys Leu His Thr 100 105 110 Ala Ile His Ile Ile Ala His Leu Phe Asn Phe Asp Cys Tyr Ser Arg 115 120 125 Ser Arg Gln Ala Thr Asp Gly Ser Leu Ala Ser Ile Leu Ser Ser Leu 130 135 140 Ser His Asp Glu Lys Lys Gly Gly Ser Trp Leu Asn Pro Ile Gln Ser 145 150 155 160 Arg Asn Thr Thr Val Glu Tyr Val Thr Phe Thr Ser Val Ala Gly Leu 165 170 175 Thr Gly Val Ile Met Thr Ile Ala Leu Ile Leu Met Val Thr Ser Ala 180 185 190 Thr Glu Phe Ile Arg Arg Ser Tyr Phe Glu Val Phe Trp Tyr Thr His 195 200 205 His Leu Phe Ile Phe Tyr Ile Leu Gly Leu Gly Ile His Gly Ile Gly 210 215 220 Gly Ile Val Arg Gly Gln Thr Glu Glu Ser Met Asn Glu Ser His Pro 225 230 235 240 Arg Lys Cys Ala Glu Ser Phe Glu Met Trp Asp Asp Arg Asp Ser His 245 250 255 Cys Arg Arg Pro Lys Phe Glu Gly His Pro Pro Glu Ser Trp Lys Trp 260 265 270 Ile Leu Ala Pro Val Ile Leu Tyr Ile Cys Glu Arg Ile Leu Arg Phe 275 280 285 Tyr Arg Ser Gln Gln Lys Val Val Ile Thr Lys Val Val Met His Pro 290 295 300 Ser Lys Val Leu Glu Leu Gln Met Asn Lys Arg Gly Phe Ser Met Glu 305 310 315 320 Val Gly Gln Tyr Ile Phe Val Asn Cys Pro Ser Ile Ser Leu Leu Glu 325 330 335 Trp His Pro Phe Thr Leu Thr Ser Ala Pro Glu Glu Asp Phe Phe Ser 340 345 350 Ile His Ile Arg Ala Ala Gly Asp Trp Thr Glu Asn Leu Ile Arg Ala 355 360 365 Phe Glu Gln Gln Tyr Ser Pro Ile Pro Arg Ile Glu Val Asp Gly Pro 370 375 380 Phe Gly Thr Ala Ser Glu Asp Val Phe Gln Tyr Glu Val Ala Val Leu 385 390 395 400 Val Gly Ala Gly Ile Gly Val Thr Pro Phe Ala Ser Ile Leu Lys Ser 405 410 415 Ile Trp Tyr Lys Phe Gln Cys Ala Asp His Asn Leu Lys Thr Lys Lys 420 425 430 Ile Tyr Phe Tyr Trp Ile Cys Arg Glu Thr Gly Ala Phe Ser Trp Phe 435 440 445 Asn Asn Leu Leu Thr Ser Leu Glu Gln Glu Met Glu Glu Leu Gly Lys 450 455 460 Val Gly Phe Leu Asn Tyr Arg Leu Phe Leu Thr Gly Trp Asp Ser Asn 465 470 475 480 Ile Val Gly His Ala Ala Leu Asn Phe Asp Lys Ala Thr Asp Ile Val 485 490 495 Thr Gly Leu Lys Gln Lys Thr Ser Phe Gly Arg Pro Met Trp Asp Asn 500 505 510 Glu Phe Ser Thr Ile Ala Thr Ser His Pro Lys Ser Val Val Gly Val 515 520 525 Phe Leu Cys Gly Pro Arg Thr Leu Ala Lys Ser Leu Arg Lys Cys Cys 530 535 540 His Arg Tyr Ser Ser Leu Asp Pro Arg Lys Val Gln Phe Tyr Phe Asn 545 550 555 560 Lys Glu Asn Phe 15 24 DNA Artificial Sequence Synthetic Primer 15 ctcattgtca cactcctcga cagc 24 16 24 DNA Artificial Sequence Synthetic Primer 16 tgggtctgat gccttgaagg actc 24 17 24 DNA Artificial Sequence Synthetic Primer 17 atcaagcggc cccctttttt tcac 24 18 24 DNA Artificial Sequence Synthetic Primer 18 ctgaacatcc ccaccattgc tcgc 24 19 24 DNA Artificial Sequence Synthetic Primer 19 ctgaacatcc ccaccattgc tcgc 24 20 24 DNA Artificial Sequence Synthetic Primer 20 gaagccgaac ttctcacaat ggcc 24 21 24 DNA Artificial Sequence Synthetic Primer 21 cctcacctct ccaagctctg cccc 24 22 24 DNA Artificial Sequence Synthetic Primer 22 ttgaacaatt ttataagatg ccgg 24 23 25 DNA Artificial Sequence Synthetic Primer 23 agaggaacac gacaatcagc cttag 25 24 25 DNA Artificial Sequence Synthetic Primer 24 ggagtttcaa gatgcgtgga aacta 25 25 31 DNA Artificial Sequence Synthetic Primer 25 gctactcgag tgtgccaatt tcacctggca t 31 26 31 DNA Artificial Sequence Synthetic Primer 26 aactctcgag tgtcaagagg tggtttggag c 31 

We claim:
 1. An isolated protein capable of stimulating superoxide production, wherein the isolated protein comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or a fragment thereof, or a conservative substitution thereof.
 2. The isolated protein or fragment thereof of claim 1, further comprising a deletion thereof, an addition thereto, or a substitution thereto of less than 20% of the amino acid sequence.
 3. The isolated protein or fragment thereof of claim 1, further comprising a deletion thereof, an addition thereto, or a substitution thereto of less than 10% of the amino acid sequence.
 4. An isolated protein capable of stimulating superoxide production, wherein the isolated protein comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, a deletion thereof or an addition thereto of no more than about 20% of the amino acid sequence, or a conservative substitution thereof, wherein the conservative substitution comprises substitution of: a) alanine, serine, or threonine for each other; b) aspartic acid or glutamic acid for each other; c) asparagine or glutamine for each other; d) arginine or lysine for each other; e) isoleucine, leucine, methionine, or valine for each other; and f) phenylalanine, tyrosine, or tryptophan for each other.
 5. The nucleotide sequence encoding for the protein, the fragment thereof or the conservative substitution thereof as recited in claim
 1. 6. The nucleotide sequence of claim 5, wherein the nucleotide sequence comprises SEQ ID NO:1, a fragment thereof, or a conservative substitution thereof.
 7. The nucleotide sequence of claim 5, wherein the nucleotide sequence comprises SEQ ID NO:3, a fragment thereof, or a conservative substitution thereof.
 8. A vector, wherein the vector comprises a nucleotide sequence encoding for the protein, the fragment thereof or the conservative substitution thereof, as recited in claim
 1. 9. The vector of claim 8 wherein the nucleotide sequence comprises SEQ ID NO:1 or SEQ ID NO:3, a fragment thereof, or a conservative substitution thereof.
 10. A cell containing the vector of claim
 8. 11. A cell containing the vector of claim
 9. 12. An antibody, wherein the antibody is capable of binding to the protein, the fragment thereof, or the conservative substitution thereof, as recited in claim
 1. 13. The antibody of claim 12, wherein the protein, the fragment thereof, or the conservative substitution thereof, has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, a fragment thereof, or a conservative substitution thereof.
 14. A method of stimulating superoxide formation comprising administration, in vitro or in vivo, of a composition comprising the protein, the fragment thereof, or the conservative substitution thereof of claim 1 in a pharmaceutically acceptable carrier.
 15. The method of claim 14, wherein the protein, the fragment thereof, or the conservative substitution thereof, comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, a fragment thereof, or a conservative substitution thereof.
 16. A method of stimulating superoxide formation comprising administration, in vitro or in vivo, of a composition comprising the vector of claim 8 in a pharmaceutically acceptable carrier.
 17. A method of stimulating superoxide formation comprising administration, in vitro or in vivo, of a composition comprising the vector of claim 9 in a pharmaceutically acceptable carrier.
 18. A method for determining the activity of a drug comprising measuring the activity of the protein, the fragment thereof or the conservative substitution thereof, as recited in claim 1, to stimulate superoxide production following administration of the drug.
 19. The method of claim 18, wherein the protein, the fragment thereof or the conservative substitution thereof comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, a fragment thereof, or a conservative substitution thereof.
 20. An isolated nucleotide sequence comprising the sequence of SEQ ID NO:5, a conservative substitution or fragment thereof.
 21. A recombinant host cell comprising the sequence of claim 20 in a reporter construct.
 22. A method for determining the activity of a drug comprising measuring the activity of the reporter construct of claim 21 to generate a protein capable of stimulating superoxide production following administration of the drug. 